Molecular markers Number:7,101,668 from the United States Patent and Trademark Office (PTO) owispatent Nucleic acid based methods for detecting the presence of E. coli or Shigella or related microorganisms in a sample using one or more E. coli or Shigella species specific nucleotide sequences are disclosed. More particularly the identification of molecules capable of binding or otherwise facilitating abnormal cell growth or abnormal physiology such as found in cancer or cellular instability is described. Further, molecular probes for performing the nucleic-acid based methods and methods of testing and selecting nucleic acid sequences suitable for same are provided. The methods and polynucleotides are useful inter alia in the testing of food and water samples, for testing for genetic and cellular instability, and for testing benign, pre-neoplastic and neoplastic disease in asymptomatic or symptomatic colorectal or gastric cancer patients or those at risk of the aforementioned conditions or those infected by Escherichieae and with other diseases or conditions.<H Molecular, markers, Molecular, marke, 7101668.html, Patent, pto, 7101668

Title: Molecular markers

Abstract: Nucleic acid based methods for detecting the presence of E. coli or Shigella or related microorganisms in a sample using one or more E. coli or Shigella species specific nucleotide sequences are disclosed. More particularly the identification of molecules capable of binding or otherwise facilitating abnormal cell growth or abnormal physiology such as found in cancer or cellular instability is described. Further, molecular probes for performing the nucleic-acid based methods and methods of testing and selecting nucleic acid sequences suitable for same are provided. The methods and polynucleotides are useful inter alia in the testing of food and water samples, for testing for genetic and cellular instability, and for testing benign, pre-neoplastic and neoplastic disease in asymptomatic or symptomatic colorectal or gastric cancer patients or those at risk of the aforementioned conditions or those infected by Escherichieae and with other diseases or conditions.

Patent Number: 7,101,668 Issued on 09/05/2006 to Ng

Inventors: Ng; Wee Chit (Singapore, SG)
Assignee: National University of Singapore (Singapore, SG)

Appl. No.: 10/240,689

Filed: April 17, 2001

PCT Filed: April 17, 2001

PCT No.: PCT/SG01/00067

371(c)(1),(2),(4) Date: September 30, 2002

PCT Pub. No.: WO01/79545

PCT Pub. Date: October 25, 2001

Current U.S. Class: 435/6

Current International Class: C12Q 1/68 (20060101)

Field of Search: 435/6,91.1,91.2 536/23.1

Foreign Patent Documents


721826	Jul., 2000	AU
0669399	Aug., 1995	EP
98/39473	Sep., 1998	WO

Other References

Blattner et al. The complete genome sequence of Escherichia coli K-12. 1997. Science vol. 277:1453-1462. cited by examiner .
Swidsinski et al. Association between intraepithelial Escherichia coli and colorectal cancer. 1998. Gastroenterology vol. 115:281-286. cited by exam- iner .
Oshima et al. DNA Research vol. 3:137-155. 1996. cited by examiner .
Buck et al. Biotechniques vol. 27:528-536. 1999. cited by examiner .
Blattner, et al., "The Complete Genome Sequence of Escherichia coli K-12", Science, 1997, vol. 277, pp. 1453-1462. cited by other .
Swidsinski, et al., "Association Between Intraepithelial Escherichia coli and Colorectal Cancer," Gastroenterology, 1998, vol. 115, pp. 281-286. cited by other.

Primary Examiner: Benzion; Gary
Assistant Examiner: Calamita; Heather G.
Attorney, Agent or Firm: Klarquist Sparkman, LLP

Claims

The invention claimed is:

1. A probe nucleotide sequence which is specific to E. coli- and/or Shigella species or related microorganism, the nucleotide sequence consisting of a sequence from SEQ ID NO: 40, the sequence from SEQ ID NO: 40 comprising at least one of the sequence consisting of nucleotides 415 to 1351, the sequence consisting of nucleotides 3151 to 4359, the sequence consisting of nucleotides 4807 to 5235, the sequence consisting of nucleotides 6073 to 7359, the sequence consisting of nucleotides 7223 to 7794, the sequence consisting of nucleotides 7278 to 7773, the sequence consisting of nucleotides 7419 to 7985, the sequence consisting of nucleotides 7562 to 7794, the sequence consisting of nucleotides 8160 to 9704 or the sequence consisting of nucleotides 9731 to 11375, and present mainly in the nucleus of cancer cells and in the normal cells adjacent to cancer cells, for the identification of a gastrointestinal cancer or tumour or a predisposition to same.

2. A method for detecting the presence of E. coli or Shigella species or related microorganisms in a sample, said method comprising subjecting said sample to genetic analysis using an E. coli- or Shigella species-specific nucleotide sequence consisting of a sequence from SEQ ID NO: 40, the sequence from SEQ ID NO: 40 comprising at least one of the sequence consisting of nucleotides 415 to 1351, the sequence consisting of nucleotides 3151 to 4359, the sequence consisting of nucleotides 4807 to 5235, the sequence consisting of nucleotides 6073 to 7359, the sequence consisting of nucleotides 7223 to 7794, the sequence consisting of nucleotides 7278 to 7773, the sequence encompassed by nucleotides 7419 to 7985, the sequence consisting of nucleotides 7562 to 7794, the sequence consisting of nucleotides 8160 to 9704 or the sequence consisting of nucleotides 9731 to 11375.

3. The method according to claim 2 wherein said genetic analysis comprises amplification of nucleotide sequence present in the sample.

4. The method according claim 2 wherein said E. coli- and/or Shigella species-specific nucleotide sequence is labeled to provide an identifiable signal.

5. The method according to 2 wherein the sample comprises a nucleic acid preparation from food, water, semi-solids or semi-liquid material, mammalian tissue, or extract or cells thereof or a nucleic acid preparation from said tissue, extract or cells.

6. The method according to claim 5 wherein the sample is mammalian tissue or extract or cells thereof.

7. The method according to claim 6 wherein the tissue, extract or cells are from a patient suffering from cancer or cellular instability or gastrointestinal infection, or a patient at risk of cancer or cellular instability.

8. The method according to claim 7 wherein the cancer is gastrointestinal cancer.

9. The method according to claim 7 wherein the cancer is colon cancer.

10. The method according to claim 7 wherein the cancer is stomach cancer.

11. The method according to claim 7 wherein the cancer is colorectal cancer.

12. An isolated nucleic acid molecule comprising the probe nucleotide sequence of claim 1 wherein said probe nucleotide sequence is capable of specifically hybridizing to E. coli- and/or Shigella species'-derived nucleic acid molecules.

13. A method of testing and selecting sequences specific to E. coli or Shigella species or related microorganisms in a sample, said method comprising subjecting a nucleic acid molecule preparation from said sample to genetic analysis using one or more E. coli or Shigella species'-specific nucleotide sequences consistig of a sequence from SEQ ID NO: 40, the sequence from SEQ ID NO: 40 comprising at least one of the sequence consisting of nucleotides 415 to 1351, the sequence consisting of nucleotides 3151 to 4359, the sequence consisting of nucleotides 4807 to 5235, the sequence consisting of nucleotides 6073 to 7359, the sequence consisting of nucleotides 7223 to 7794, the sequence consisting of nucleotides 7278 to 7773, the sequence consisting of nucleotides 7419 to 7985, the sequence consisting of nucleotides 7562 to 7794, the sequence consisting of nucleotides 8160 to 9704 or the sequence consisting of nucleotides 9731 to 11375.

14. The method according to claim 13 wherein said genetic analysis comprises amplification of nucleotide sequence present in the sample.

15. The method according to claim 13 wherein said E. coli- and/or Shigella species-specific nucleotide sequence is labeled to provide an identifiable signal.

16. The method according to claim 13 wherein the sample comprises mammalian tissue or extract or cells thereof.

17. The method according to claim 16 wherein the tissue, extract or cells are from a patient suffering from cancer or cellular instability or gastrointestinal infection, or a patient at risk of cancer or cellular instability.

18. The method according to claim 17 wherein the cancer is gastrointestinal cancer.

19. The method according to claim 17 wherein the cancer is colon cancer.

20. The method according to claim 17 wherein the cancer is stomach cancer.

21. The method according to claim 17 wherein the cancer is colorectal cancer.

22. A nucleotide sequence identified by the method according to claim 13 wherein said sequence is capable of specifically hybridizing to E. coli and/or Shigella species'-derived nucleic acid molecules.

Description

FIELD OF THE INVENTION

The present invention relates generally to nucleic acid based methods for detecting the presence of E. coli or Shigella or related microorganisms in a sample using one or more E. coli or Shigella species specific nucleotide sequences. More particularly, the present invention permits the identification of molecules capable of binding or otherwise facilitating abnormal cell growth or abnormal physiology such as found in cancer or cellular instability. The present invention further provides molecular probes for performing the nucleic-acid based methods of the invention and methods of testing and selecting nucleic acid sequences suitable for same. The methods and polynucleotides of the present invention are useful inter alia in the testing of food and water samples, for testing for genetic and cellular instability, and for testing for benign, pre-neoplastic and neoplastic disease in asymptomatic or symptomatic colorectal or gastric cancer patients or those at risk of the aforementioned conditions or those infected by Escherichieae and with other diseases or conditions.

BACKGROUND OF THE INVENTION

The identification of bacteria can be carried out using biochemical, cultural, antibody recognition and molecular biological tests (Feng P C S and Hartman P A: Fluorogenic Assays for Immediate Confirmation of Escherichia coli. 1982. Falkow S, Habermehl K O. ed: Rapid Methods and Automation in Microbiology and Immunology. Springer-Verlag, Berlin 1985: 30 33. AOAC Official Methods of Analysis 1995. Pepper Ill., Gerba C P and Brendecke J W: Environmental Microbiology. A laboratory Manual. Academic Press 1995.)

Food and Water Hygiene

Biochemical Test and Culture Medium

The most probable number (MPN) is the common method for the detection and quantitation of E. coli in foods. This method detects E. coli on the basis of the bacteria's ability to ferment lactose with the evolution of gas. Other non-E. coli organisms also ferment lactose and, therefore, several selective enrichment steps are required in order to sequentially select for coliform bacteria and E. coli.

This widely used MPN method has several limitations. Many clinical E. coli isolates are lactose negative and thus are not detected using the MPN method. The MPN method requires a minimum of about four days to determine the absence of E. coli in food products and about seven days are required to get confirmed results. The growth of some E. coli, including the serotype 0157:H7 strains, is severely inhibited by the selectivity of the EC broth at 45.5.degree. C. and gas production in the MPN method is susceptible to interference by high levels of competitor organisms.

More rapid methods for detecting E. coli are needed because of the time and accuracy limitations of the MPN method. It has been reported that 94% to 97% of E. coli strains possess the B-D-glucuronidase that can be detected by specific hydrolysis of a synthetic substrate, 4-methylumbelliferyl-B-D-glucuronide (MUG), to a fluorescent end product. When MUG is incorporated into lauryl sulfate tryptose (LST) broth, 10.sup.7 to 10.sup.8 CFU/ml of E. coli will yield this fluorescent product which can be detected under longwave UV light. However, a number of enteropathogenic E. coli including serotype 0157:H7 strains, do not possess the B-D-glucuronidase enzyme, do not exhibit fluorescence in LST-MUG medium, and therefore yield false-negative results using the MUG method. In addition, the selectivity of the method is compromised by the fact that some Shigella, Citrobacter, Ecterobacter, Klebsiella, Salmonella, and Yersinia species also produce B-D-glucuronidase and therefore yield false-positive results.

Another widely used test, the Analytical profile index (API) test strips, produced by BioMerieux (France), may be used to obtain test results quickly. These consist of a series of miniature capsules on molded plastic strips, each of which contains a sterile dehydrated medium in powder form. Addition of water containing a bacterial suspension simultaneously re-hydrates and inoculates the medium. A rapid reaction is obtained because of the small volume of medium and the large inoculum used. The identification of the unknown bacterium is achieved by determining a seven digit profile index number and consulting the API profile recognition system. However, there are strains of E. coli that yield a low discrimination value with the API strips.

When this occurs, further identification with sugar test is required for affirmation. Acid production from sugars such as D-Adonitol, Cellobiose, Lactose and D-Xylose are additional biochemical test for differentiation of Escherichia species and related species.

DNA Probes

The use of genetic probes in the detection of microorganisms is popular because they obviate the need for pure cultures, and are specific, sensitive, fast and reliable (Fred C. Tenover: DNA probes for infectious diseases. CRC Press, Inc. 1989). In DNA probe test, it is essential to know something about the nucleotide sequence of the microorganisms under investigation.

Bacteria belonging to different families or strains can be differentiated on the basis of heterogeneity in genetic sequences. One approach is the identification and use of specific toxin genes of disease causing strains to distinguish them from the normal flora. Another approach makes use of the conserved and polymorphic sites that are found in bacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. The combination of the polymerase chain reaction technique for gene amplification, followed by sequencing of polymorphic regions and phylogenetic analysis of the resulting sequence information can also assist in strain identification. (Relman et al. The New Engl J of Medicine, 327: 293 301, 1992, Kui et al. FEMS Microbiology Letters 57:19 24, 1989. DeLong et al. Science 243: 1360 1363, 1989).

The E. coli identification kit produced by gene-trak systems, Framingham, Mass., USA, uses DNA oligonucleotides that complement the 16S rRNA. This assay uses hybridization techniques to detect E. coli, non-coli Escherichia fergusonii and Shigella species.

Another way of identifying bacteria specific DNA probes is by using randomly cloned chromosomal fragments. This involves the cloning of restriction enzyme cleaved genomic DNA of a bacteria, and selection of specific clones by determining their hybridization profiles by hybridization against its own species-sequences and other species-sequences. Only clones that hybridize to sequences from the same species but the clones were derived from will be selected (Tenover FC: DNA Probes for Infectious Diseases. CRC Press 1989).

Gastrointestinal Infection

Colorectal cancer is one of the top three cancer killers in the world. Factors implicated in its etiology include inappropriate diet, environmental factors and lack of reliable diagnostic markers. Recently, greater understanding of the genetic predisposition to colon cancer has been achieved through the identification of genes responsible for such susceptibility (Cowell J K, ed: In Molecular Genetics of Cancer. Dunlop M G: Molecular genetics of colon cancer. 1995. 113 134). Despite intensive research efforts, the mortality rate from colorectal cancer has not declined dramatically over the last 40 years.

Markers associated with cancer initiation or progression are important in patient care. Tumours diagnosed at an early stage can usually be cured by surgical excision or polypectomy (surgical excision cures 90% of patients with adenoma or carcinomas that are confined to the mucosa). Patients with advanced disease have a poor prognosis as mortality increases to more than 90% after metastasis takes place.

The gastrointestinal tract is often exposed to a range of microorganisms. When bacteria come into contact with a susceptible host, they can establish either a transient presence, colonize the individual, infect the individual or evolve with the host. The outcome can either be harmless, acute illness or a chronic condition that may lead to a serious outcome (Gibson G R and Macfarlane G T: Human Colonic Bacteria: Role in Nutrition, Physiology, and Pathology. CRC Press, Inc., 1995).

Bacteria have been associated with inflammatory bowel disease such as ulcerative colitis and Crohn's disease (Giaffer et al. Gut 33:646 650, 1992, Cartun et al. Mod Pathol 6:212 219, 1993; Liu et al. Gastroenterology 108:1396 404, 1995). In addition, patients with pan-colitis of long duration are at risk of developing colorectal cancer (Wanebo H J: In Colorectal Cancer. Lev R: Precursors of Colon Carcinoma 1993; 158 163). Although frequently implicated, the role of bacteria in colon related disease remains ill-defined and controversial. The identification of bacteria in physical proximity to diseased tissue does not provide definitive proof of a causal relationship between a bacterium and the diseased condition. This is especially so when the bacteria are commonly found surrounding the tissue (Swidsinski et al. Gastroenterology 115:281 286, 1998), as is the case in the colon, and there is no additional information to differentiate between bacteria. It is perhaps more convincing if the bacterium can be shown to be positioned in-situ in the diseased tissue and when isolated and characterized found to possess properties that will substantiate its presence within the tissue.

The bacterium Helicobacter pylori is an accepted Group 1 (definite) biological carcinogen for gastric cancer and causes of related gastric conditions such as duodenal ulcer, gastric ulcer and ulcer complications. H. pylori attaches to and thrives on the gastric mucosa resulting in a chronic immunological response from the host. (Marshall, B. J. Gastroenterologist 1:241 247, 1993). It is not firmly established whether H. pylori has invasive properties. However, pathogenic strains have been identified that can cause epithelial cell damage and mucosal ulceration on an intragastric administration to mice (Telford et al. J Exp Med 179:1653 1658, 1994) The question remains whether H. pylori is the only important factor in the development of gastric cancer because of its high infection/disease ratio. The current consensus is that there may be other factors other than H. pylori infection that are also important in gastric cancer risk (National Institutes of Health Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease 1994). A separate study put forward the theory that a synergistic interaction between a non-invasive bacteria and other enteropathogens can facilitate invasion by the otherwise non-invasive bacteria (Geir Bukhowm and Georg Kapperud, Infection and Immunity 55:2816 2821, 1987).

Numerous in-vivo and in-vitro studies have vividly shown that microorganism carry transmissible tumorigenic genetic information. Mutagenesis in such instances is either by transposition or site-specific recombination facilitated by conjugation, transformation and transduction. This information is constantly being exploited scientifically in creating mutants (Sherratt D J (ed): Mobile genetic elements. Dale J W: Molecular genetics of bacteria. 2.sup.nd Edition. John Wiley and Sons Ltd. Oxford University Press 1995). In 1995, Couralin et al. showed that invasive strains of Shigella flexneri and E. coli can carry out gene transfer that are stably inherited and expressed by the mammalian cell progeny (Courralin et al., C. R. Acad. Sci. Paris 318:1207 1212, 1995). Therefore, it is quite possible that the persistent presence of bacterial genetic sequences in the nucleus of mammalian cells can lead to genetic instability that may ultimately give rise to a tumour cell.

Bacterial invasion can stimulate similar a pattern of protein phosphorylation to that induced by growth factor (e.g. EGF) and cellular proliferative responses may then be altered with consequences for disease progression. (Galan et al. Nature 357:588 589, 1992). In addition, bacterial disruption of cell-cell interaction may affect cell proliferation patterns and differentiation (Epenetos A A and Pignatelli M (ed): Cell Adhesion Molecules in Cancer and Inflammation; Pignatelli et al.: Adhesion molecules in neoplasia: An overview. Chapter 1:1 13. Harwood academic publishers 1995). Cytonecrotizing factors have been identified that can cause formation of large multinucleated cells and cells spreading in tissue cultures. (Denko et al. Experimental Cell Research 234:132 138, 1997; Lemichez et al., Molec Microbiol 24:1061 1070, 1997; Machesky, L. M. and Hall, A, TICB 6:304 310, 1996). Accordingly, the persistence presence of bacteria can cause cellular changes leading to cell disorientation, proliferation and changes in cell morphology.

One cancer causing effect of bacteria is when Agrobacterium tumefaciens, a soil phytopathogen, genetically transforms plant cells by the transfer of the tumour-inducing (Ti) plasmid to the plant genome where its integration and expression result in the crown gall phenotype. A crown gall is a tumorous proliferation of plant cells which are released from normal metabolic and reproductive controls (Hughes M A: Plant Molecular Genetics. Addison Wesley Longman Ltd. 1996).

People travelling across continents may suffer from traveler's diarrhoea as the bacteria they are exposed to are not common in their county. The assays/kits that are used for detecting microorganisms in the Asia-Pacific region are imported from other continents and these imported assays/kits may not be as sensitive or as specific for the bacteria in the Asia-Pacific region.

Microorganisms transmitted by water and food usually grow in the intestinal tract of man and animals and leave the body in the faeces. Bacteria are known to possess gene sequences that make them toxigenic, hemorrhagic, invasive and adherent to tissues. Acute bacterial infection is well documented but it is still not known that if bacteria that do not cause overt symptoms but persist and remain undetected in their host can cause diseases with time. Therefore, it is important that the assays that are available are sensitive and specific for a wide range of pathogens.

The E. coli genetic sequence is published. (Blattner et al. Science 277:1453 1474, 1997). Some of its genetic sequence has homology to other bacteria (Janda J M and Abbott S L: The Enterobacteria. Lippincott-Raven Press 1998). The inventor, in accordance with the present invention, has identified E. coli DNA sequences which are unique to the Escherichae family and furthermore has shown that biochemical and cultural tests presently available are not adequate for detecting this family of bacteria. The present polynucleotide sequence in the genome of strains of the Escherichieae genus (Escherichia and Shigella), have proven to be more informative than the agar plates EMB, MacConkey and MUG. They can be used to detect E. coli that is either EMB, MacConkey or MUG negative. The sequences are also found in 0157:H7 and 029:NM strains of E. coli. Therefore, the present molecular markers provide improved tools for the detection and characterization of E. coli.

In addition, the invention permits the use of the sequence(s) to study the outcome of tissue infection in-situ. The present gene sequences are more specific than the gene-trak sequence (gene-trak systems) and the sequences can be amplified many-fold to increase their detection limit. This makes the present invention useful for studying the role of microorganism in gastrointestinal and other disease conditions. The presence of the polynucleotide sequence in cells can be located by the use of the polymerase-chain-reaction amplification technique in-situ followed by hybridization to the in-situ amplified signals with sequence specific DNA probe.

The identification of these specific polynucleotide sequence(s) that can be used to detect for the presence of strains of E. coli and Shigella and related microorganisms in food, water, fecal specimens, tissues, secretions and other biological, environmental and/or laboratory samples is important for health reasons as it enables one to check on the quality of food and water hygiene and monitor transmission of the microorganism. Sensitive detection techniques and methods for assessing the role of bacteria in clinical conditions will ultimately help in the control of harmful microorganisms.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

One aspect of the present invention provides a new use for the whole of Formula I and sequences within Formula I, as markers for species of bacteria within the Escherichieae family specifically Escherichia coli and Shigella species or related microorganisms.

Another aspect of the present invention relates to the use of the polynucleotide sequence of formula I to generate gene probes of smaller size which singularly or in combination have specificity for E. coli strains or related microorganisms but not necessarily specific for all the Shigella species.

A further aspect of the present invention provides Formula I and the smaller gene sequences within it as a means to detect the presence, in liquids, semisolids and solids combinations thereof or in aerosols or gases, of species of bacteria within the Escherichieae family specifically E. coli and some or all of the Shigella species so that high standard of sanitation can be achieved.

Yet another aspect of the present invention provides the aforementioned sequence(s) as a means to detect infection in a sample and/or a combination of samples by members of the Escherichieae family as aforementioned. Samples are defined in this invention as tissues or cells or explants of either human, animals or plant origin, such given examples being tissue/cells found in the colon, stomach, and other parts of the human or animal anatomy as well as in food, industrial and/or environmental samples.

A further aspect of the present invention provides a method for testing and identifying the various genes within formula I as new means to detect for changes in DNA content in cells infected or previously infected with the aforesaid Escherichieae family of bacteria (E. coli and Shigella species or related microorganisms). A cell is defined in this invention as a cell found in the animal and plant kingdom. Changes in DNA content in a cell in accordance with this invention includes DNA sequences found in the cell which differs by one or more nucleotide substitutions, additions and/or deletions of existing DNA or by the introduction of a heterologous DNA.

Yet another aspect of the present invention provides the aforementioned Formula I within which polynucleotide sequences are a marker for use in recognizing early cellular DNA changes associated with any one or more members of the Escherichieae family (E. coli and Shigella species and related microorganisms) in the colonic epithelium before the histology criteria for such cellular changes are detectable. Early changes are defined in this invention by the presence of at least bacterial DNA sequences that are present in high, low copy numbers or present as single copies per haploid genome in a normal population.

Still yet another aspect of the present invention provides the aforementioned Formula I within which polynucleotide sequences are a marker for use in recognizing pre-malignant changes associated with any one or more members of the Escherichieae family (E. coli and Shigella species and related microorganisms) in the colonic epithelium as defined by histology criteria for such pre-malignant tissue. Pre-malignant changes are defined in this invention by the presence of at least bacterial DNA sequences that are present in high or low copy numbers or present as single copies per haploid genome in a normal population and are supported by histology criteria.

Even still another aspect of the present invention provides the aforementioned Formula I within which polynucleotide sequences can be a marker for use in recognizing malignant changes associated with any one or more members of the Escherichieae family (E. coli and Shigella species and related microorganism) in the colonic epithelium and malignant colonic tumours residing in other tissues. Malignant changes are defined by histology criteria. Malignant changes associated with any one member of the Escherichieae family are defined in this invention by the presence of at least bacterial DNA sequences that are present in high or low copy numbers or present as single copies per haploid genome in a normal population.

Another, aspect of the present invention provides the aforementioned formula I within which polynucleotide sequences can be used as a marker for detecting pre-malignant changes associated with any one or more members of the Escherichieae family in the gastric mucosa as defined by histology criteria for such pre-malignant tissues. Pre-malignant changes are defined in this invention by the presence of at least bacteria DNA sequences that are present in high or low copy numbers or present as single copies per haploid genome in a normal population and its histology criteria for the tissue defined.

Yet another aspect of the present invention provides the aforementioned Formula I sequence within which polynucleotide sequences can be used as a marker to recognize malignant changes associated with any one or more members of the Escherichieae family in the malignant gastric tumours and malignant gastric tumours residing in other tissues. Malignant changes are defined inter alia by histology criteria. Malignant changes associated with the Escherichieae family in this invention is defined by the present of at least DNA sequences that are present in high or low copy numbers or present as single copies per haploid genome in a normal population.

Still another aspect of the present invention provides the aforementioned Formula I within which sequences can be used as markers to recognize patients that are found harboring any one or more member of the Escherichieae family relative to normal patients not haboring the same and are thus identified as marker of infection of said family that are important in patient care.

Even still another aspect of the present invention provides the aforementioned Formula I sequence within which polynucleotide sequences can be used as markers being found in colorectal cancer patients relative to normal patients and thus identified as a marker of malignant disease that is important in patient care.

Even yet another aspect of the present invention provides the aforementioned marker that is found in gastric cancer patients relative to normal patients and is thus identified as a marker of malignant disease that is important in patient care.

Another aspect of the present invention provides a marker for cellular instability and therefore a marker for predisposition to cellular carcinogenesis. Cellular instability may occur as a forerunner to cellular carcinogenesis or other condition and is characterized herein by changes in DNA content comprising one or more nucleotide substitutions, additions and/or deletions of existing DNA or by the presence of heterologous DNA.

Another aspect of the present invention provides a method of testing and selecting sequences in E. coli and Shigella species and related microorganisms as markers for use to detect changes in DNA content in cells in order to recognize cellular instability and, therefore, predisposition to cellular carcinogenesis, predisposition to colon and gastric cancer and as markers for use in recognizing benign, pre-malignant and malignant gastrointestinal tissues as optionally defined by histology criteria.

In accordance with the present invention, it is shown that Formula I comprises polynucleotide DNA sequence marker(s) for the Escherichieae family specifically E. coli species and Shigella species or related microorganisms. This Formula I and the various genes and sequence it contains allow the differentiation of the aforementioned members of the Escherichieae family from other bacteria families. In addition, the presence of such bacteria as indicated by the presence of the DNA sequences allows study of sanitation and health related matters such as infection, predisposition to cancer, cancer and cell instability.

Reference to "related microorganisms" includes microorganisms which are related at the immunological, biochemical, disease-causing, physiological or genetic levels. A derivative or mutant form of E. coli or Shigella species is an example of a related microorganism.

The present invention furthermore provides a method of testing and selecting other sequences in E. coli and Shigella species and related microorganisms as markers to test for their presence in cells with abnormal cell growth or physiology associated with cancer or a predisposition to the development of cancer.

Yet still another aspect of the present invention relates to a new use for the various polynucleotide sequences within Formula I as molecular probes in the determination of whether samples contain members of the Escherichieae family such as E. coli and Shigella species or related microorganisms.

In another aspect, the present invention provides methods for enhancement in the specificity and sensitivity of detecting the presence, among other bacteria of E. coli species and some of the Shigella species with some of the aforementioned sequences.

The presence of the polynucleotides sequences in food and water is evidence that they are contaminated with members of Escherichieae family such as E. coli species and probably some or all of the Shigella species. Thus the present molecular probes provide an alternative to microbiological and biochemical assays which are less specific, sensitive, reliable, often required for pure cultures, and are more time consuming.

A further related aspect of the present invention provides a new use for the sequences within the Formula I for determining whether tissue samples contain the DNA markers that originate from members of the E. coli and Shigella species. Both species within this family are known to have invasive, adherent and toxigenic properties. This aspect relates to the new use of polynucleotide sequence(s) within Formula I as marker(s) for detecting infection by identifying samples such as, for example, colonic and gastric mucosa tissues that contain them. The presence of the polynucleotide sequences in tissues is evident that the tissues are infected by members of the E. coli and Shigella species.

A further aspect of the instant invention relates to a new use for the sequences within the Formula I for determining which cell type within tissues samples contain marker DNA sequences that originate from members of the Escherichieae family such as E. coli and the Shigella species and related microorganisms.

A further aspect of the instant invention relates to a new use for the aforementioned polynucleotide sequence(s) within Formula I as a maker(s) for detecting changes in cellular DNA composition in, for example, colonic and gastric mucosa cells before histology criteria for changes are detectable. The presence of the polynucleotide sequences in cells of tissues is evidence that the cells are infected by members of the E. coli and Shigella species. Changes in cellular DNA composition are defined in this particular aspect of the invention by the presence of at least bacteria DNA sequences that are present in high or low copy numbers or present as single copies per haploid genome in a normal population. The polynucleotide sequence of the present invention are only found in Escherichieae family and, therefore, their presence in other species such as in eukaryotic cells, for an example, is a sign of an abnormal event. Accordingly, the present invention provides one or more molecular marker for screening patients to identify those who are at risk of having gastrointestinal tumours (benign, pre-malignant, or malignant).

A further additional aspect of the present invention relates to a new use for the sequences within the Formula I for determining whether pre-malignant tumours as defined by histology criteria contain aforementioned polynucleotides sequences that originate from members of the E. coli and Shigella species or related microorganisms. This aspect relates to the new use of the marker for detecting the presence of any one or more member of the Escherichieae family in the pre-malignant tumours such as colonic and gastric tissues as defined by histology criteria. The presence of the polynucleotide sequence in the cells of pre-malignant colonic and gastric tumours is evidence that the cells are infected by or contain DNA sequences of members of the E. coli and Shigella species. These pre-malignant tumours contain the presence of at least bacteria DNA sequences that are present in high or low copy numbers or present as single copies per haploid genome in a normal population. The polynucleotide sequence of the present invention are only found in Escherichieae family and, therefore, their presence in other species such as in eukaryotic cells, for an example, is a sign of an abnormal event. Accordingly, the present invention provides one or more molecular markers for screening patients to identify those at risk of having gastrointestinal tumours (benign, pre-malignant, malignant).

The present invention also relates to a new use for the aforementioned polynucleotide sequence(s) as a marker for determining whether malignant changes in the colonic and gastric mucosa contain marker sequences that originate from members of the E. coli and Shigella species. Malignant changes are defined by conventional histological criteria. This aspect of the invention relates to the new use of the marker for detecting the DNA presence of any one or more member of the Escherichieae family in the malignant tumours. The presence of the polynucleotide sequence in the cells of these malignant tumours is evidence that the cells are infected by or contain DNA sequences of members of the E. coli and/or Shigella species. These malignant tumours contain the presence of at least bacteria DNA sequences that are present in high, low copy numbers or present as single copies per haploid genome in a normal population. The polynucleotide sequence of the present invention are only found in Escherichieae family and, therefore, their presence in other species such as in eukaryotic cells, for an example, is a sign of an abnormal event. Accordingly, the present invention provides one or more molecular markers for screening patients having gastrointestinal tumours (benign, pre-malignant, malignant).

The instant invention provides in a related embodiment a new use for the aforementioned polynucleotide DNA sequence(s) as markers for determining the presence of any member of the E. coli and Shigella species in metastatic cells of colonic or of gastric tumour origin residing in other tissues. This aspect of the invention relates to the new use of the marker sequence for detecting the DNA presence of any one or more member of the E. coli and/or Shigella species in the metastatic cells. The presence of the polynucleotide sequence in the cells of these malignant tumours is evidence that the cells are infected by or contain DNA sequences of members of the E. coli and/or Shigella species. These metastatic cells contain the presence of at least bacteria DNA sequences that are present m high or low copy numbers or present as single copies per haploid genome in a normal population. The polynucleotide sequence of the present invention are only found in Escherichieae family and, therefore, their presence in other species such as in eukaryotic cells, for an example, is a sign of an abnormal event. Accordingly, the present invention provides one or more molecular markers for screening patients having gastrointestinal metastatic cells.

The present invention furthermore relates in a different aspect to a new use for the formula I sequence that is found in E. coli and Shigella species or in related microorganisms, as a marker for determining cells that possess it. The invention relates to the new use of the marker sequence for detecting the DNA presence of any one or more member of the E. coli and/or Shigella species in the cells. This gene sequence is only found in Escherichieae family and therefore its presence in high, low copy numbers or as single copies per haploid genome in a normal population in eukaryotic cells, for an example, is a sign of an abnormal event that may lead to genetic instability of cell that possess it. It provides as a molecular marker for risk of and genetic instability and therefore tumourigenesis.

Even more particularly the present invention, in one aspect, provides a method for detecting the presence of E. coli or Shigella species or related microorganisms in a sample, said method comprising subjecting a nucleic acid molecule preparation from said sample to genetic analysis using one or more E. coli- or Shigella species'-specific nucleotide sequences obtainable from one or more nucleotide sequences of Formula 1 and/or Table 1 wherein the ability for said E. coli- or Shigella species'-specific nucleotide sequences to hybridize to complementary nucleotide sequences in the nucleic acid preparation is indicative of the presence of E. coli, Shigella species or related microorganisms.

In a further aspect of the present invention there is provided a method for detecting the presence of E. coli and/or Shigella species or related microorganisms in a sample as hereinbefore described wherein the nucleotide sequences of Formula I comprises from nucleotide position 246 of GenBank Accession No. AE000201 to nucleotide position 6693 of GenBank Accession No. AE000203 including the nucleotide sequence of GenBank Accession No. AE000202.

Still a further aspect of the present invention provides a method for detecting the presence of E. coli or Shigella species or related microorganisms in a sample as hereinbefore described wherein the E. coli- and/or Shigella species'-specific nucleotide sequences comprises at least 8 nucleotides in length.

A related aspect of the present invention discloses a method for detecting the presence of E. coli or Shigella species or related microorganisms in a sample as hereinbefore described wherein hybridization of E. coli- and/or Shigella species'-specific nucleotide sequences to the nucleic acid preparation is detected by the presence of amplified nucleic acid products.

A further related aspect of the present invention provides a method for detecting the presence of E. coli, Shigella species or related microorganisms in a sample wherein hybridization of E. coli- and/or Shigella species'-specific nucleotide sequences to the nucleic acid preparation or the presence of amplified nucleic acid products is detected by a reporter molecule giving an identifiable signal.

Still yet a further related aspect of the present invention provides a method for detecting the presence of E. coli, Shigella species or related microorganisms in a the sample wherein the sample comprises food, water, semi-solids or semi-liquid material, mammalian tissue, tissue extract or cells of tissue or normal tissue or tissue predisposed to cancer growth or malignancy or cellular instability.

In a particularly preferred aspect of the present the mammalian tissue is associated with colon, stomach or colorectal tissue.

A related aspect of the present invention provides a method for identifying nucleotide sequences, or their expressed products, capable of inducing or otherwise facilitating abnormal cell growth or abnormal physiology, said method comprising introducing a nucleotide sequence comprising E. coli- and/or Shigella species'-specific nucleotide sequences from the nucleotide sequences in Formula I into cells and observing morphological and/or physiological changes to said cells compared to control cells without said introduced nucleotide sequences wherein the presence of abnormal morphology and/or physiology in a cell is indicative of a nucleotide sequence from Formula I, or a polypeptide expressed therefrom, which is capable of inducing or facilitating abnormal cell growth or physiology.

In a further preferred aspect of the instant invention the abnormal cell growth or physiology is associated with cancer or a predisposition to the development of cancer or cellular instability.

A further aspect of the present invention provides a molecular probe comprising at least 8 nucleotides obtainable from the nucleotide sequences of Formula I wherein said molecular probe is capable of specifically hybridizing to E. coli- and/or Shigella species'-derived nucleic acid molecules.

Yet more particularly the present invention encompasses a use of a nucleotide sequence obtainable from the nucleotide sequence of Formula I in the manufacture of a molecular probe for the identification of E. coli and/or Shigella species and/or for the identification of a cellular instability or a cancer or tumor or a predisposition to development of same.

Still even yet more especially the present invention provides a method for testing and selecting other sequences in E. coli, Shigella species or related microorganisms in a sample, said method comprising subjecting a nucleic acid molecule preparation from said sample to genetic analysis using one or more E. coli or Shigella species'-specific nucleotide sequences obtainable from one or more nucleotide sequences of Formula 1 and/or Table 1 wherein the ability for said E. coli- or Shigella species'-specific nucleotide sequences to hybridize to complementary nucleotide sequences in the nucleic acid preparation is indicative of an E. coli or Shigella species'-specific nucleotide sequence.

Another aspect of the instant invention provides a molecular probe of at least 8 nucleotides, identified by the methods as herein described wherein said probe comprises a sequence of nucleotides from Formula I and wherein said molecular probe is capable of specifically hybridizing to E. coli and/or Shigella species'-derived nucleic acids.

The invention still yet provides the use of a nucleotide sequence identified by the methods herein disclosed in the manufacture of a molecular probe for the identification of E. coli, Shigella species and/or for the identification of a cellular instability or a cancer or tumour or a predisposition to development of same.

A related aspect of the instant invention discloses a use of a nucleotide sequence specific to E. coli and/or Shigella species and/or related microorganism in the manufacture of a molecular probe for the identification of one or more gastrointestinal cancers or tumours or a predisposition to the development of same.

A final preferred aspect of the instant invention provides a molecular probe comprising a nucleotide sequence specific to E. coli and/or Shigella species and/or related micro-organism for the identification of one or more gastrointestinal cancers or tumours or a predisposition to same.

Other aspects, features and advantages of the present invention will become apparent from the detailed description that follows, or may be learned by practice of the invention.

For the sale of brevity, reference to specific microorganisms such as Escherichia coli (E. coli) or Shigella species includes reference to related microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures serve to further explain the principles of the instant invention. It is to be understood, however, that the figures are designed for purposes of illustration only, and not as a definition of the limits of the invention for which reference should be made to the claims appearing at the end of the description.

FIGS. 1a c. Schematic diagram of the various locations of the genes tested that is within the polynucleotide sequence of formula I. The formula I sequence extends from nucleotide position (nt) 246 of GenBank accession #AE000201, including sequence of GenBank accession #AE000202 to nucleotide position 6693 of GenBank accession #AE000203.

FIG. 2. Autoradiograph result of probe A hybridizing to a panel of bacteria DNA as listed in Table 2a grid C. Probe A consists of fragments 1,2,3 and 4 as depicted in FIG. 1, a c. Each fragment is generated by primer directed PCR carried out on K12 E. coli DNA and subsequently combined for .sup.32P labeling and hybridization. The gene sequence spans between nucleotide position 1163 of AE000201 through AE000202 to 503 of AE000203. The primer pairs used are: ECM-1163, torT-5750 (fragment 1, AE000201); torT-5129 AE000201, CD-1351 AE000202 (fragment 2); CD-415, ycdG 7359 (fragment 3, AE000202); ycdG-6073 AE000202, New2-503 AE000203 (fragment 4). Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 3. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid A. The gene sequence spans between nucleotide position 246 to 850 of AE000201. ECM-246 and ECM-850 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM NA pyrophosphate at 65.degree. C.

FIG. 4. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 1163 to 1958 of AE000201. ECM-1163 and ECM-1958 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 5. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid A. The gene sequence spans between nucleotide position 7218 to 7761 of AE000201. Primers tor C-7218 and tor C-7761 are used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 6. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid A. The gene sequence spans between nucleotide position 8332 to 8891 of AE000201. Primers tor A-8332 and tor A-8891 are used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 7. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 10574 to 11160 of AE000201. Primers tor D-10574 and tor D-11160 are used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 0.1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 8. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid A. The gene sequence spans between nucleotide position 415 to 1351 of AE000202. CD-415 and CD-1351 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 0.1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 9. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 3151 to 4359 of AE000202. Primers agp-3151 and agp4359 are used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 10. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid C. The gene sequence spans between nucleotide position 4807 to 5235 of AE000202. Wrb-4807 and Wrb-5235 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 11. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid A. The gene sequence spans between nucleotide position 6073 to 7359 of AE000202. Primers ycdG-6073 and ycdG-7359 are used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 0.1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 12. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid A. The gene sequence spans between nucleotide position 7223 to 7794 of AE000202. 81B-7223 and 81B-7794 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 13. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 7278 to 7773 of AE000202. 81B-7278 and 81B-7754 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 14. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid C. The gene sequence spans between nucleotide position 7419 to 7985 of AE000202. OH-7419 and OH-7985 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 15. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 7562 to 7794 of AE000202. OH-7562 and 81B-7794 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 16. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid C. The gene sequence spans between nucleotide position 8160 to 9704 of AE000202. New1-8160 and New1-9704 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 MM Na pyrophosphate at 65.degree. C.

FIG. 17. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 9731 to 11375 of AE000202. New2-9731 and B-11375 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 5.times.SSC, 0.05% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 18. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid C. The gene sequence spans between nucleotide position 9731 of AE000202 to 503 of AE000203. New 2-9731 and New2-503 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 19. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid B. The gene sequence spans between nucleotide position 5944 to 6693 of AE000203. Primers putP-5944 and putP-6693 are used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 MM Na pyrophosphate at 65.degree. C.

FIG. 20. Autoradiograph result of radiolabeled probe A hybridized to Enterobacter cloacae and K12 E. coli genomic DNA as depicted in Table 2a grid E. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 21. Autoradiograph result of radiolabeled gene probe hybridized to Enterobacter cloacae and K12 E. coli genomic DNA as depicted in Table 2a grid E. The gene sequence spans between nucleotide position 7562 to 7794 of AE000202. OH-7562 and 81B-7794 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 22. Autoradiograph result of radiolabeled gene probe hybridized to Enterobacter cloacae and K12 E. coli genomic DNA as depicted in Table 2a grid E. The gene sequence spans between nucleotide position 7223 to 7794 of AE000202. 81B-7223 and 81B-7794 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 23. Autoradiograph result of radiolabeled gene probe hybridized to bacteria DNA as listed in Table 2a grid D. The gene sequence spans between nucleotide position 7278 to 7773 of AE000202. 81B-7278 and 81B-7754 are the primers used to generate the gene probe by PCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot. Post hybridization wash condition is 1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 24 Autoradiograph result of .sup.32P radiolabeled H. pylori ribosomal gene probe hybridized to bacteria DNA as listed in Table 2a grid A. Primer pairs indicated are used for PCR amplification of H. pylori genomic DNA to generate the required gene segment. Five hundred nanogram of genomic DNA is loaded per dot. Post hybridization wash condition is 0.1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 25. Autoradiograph result of .sup.32P radiolabeled H. pylori ribosomal gene probe hybridized to bacteria DNA as listed in Table 2a grid D. Primer pairs indicated are used for PCR amplification of H. pylori genomic DNA to generate the required gene segment. Five hundred nanogram of genomic DNA is loaded per dot. Post hybridization wash condition is 0.1.times.SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C.

FIG. 26. Autoradiograph result of in vitro simulated PCRISH. H. pylori .sup.32P radiolabeled ribosomal gene probe is hybridized to products generated from its primer directed PCR amplification of H. pylori and E. coli genomic DNA and total DNA of H. pylori and E. coli isolates obtained from patients' fecal specimens. The post hybridization wash condition is 5.times.SSC, 0.05% w/v SDS, 20 mM Na pyrophosphate at 65.degree. C. See Table 2a grid F.

Table 1. Oligonucleotide Primers.

Table 2a. Grids A,B,C show the different types of bacteria genomic DNA loaded onto corresponding nylon plus membrane and hybridized to random primed .sup.32P-radiolabeled gene probes. Columns W to Y for all three grids have the same panel of bacteria DNA. Column Z as indicated, has only a few bacteria DNA that is common among them. DNA of E. coli isolates obtained from patient fecal specimens are: 219/1, 196/1, 196/28, 197/5, 218/40, 142/31, 179/36, and 117/3B. Patient's Shigella sonnei isolate is 219/1. 078:H11 and 0157:H7 are commercial E. coli strains. TG2 is a gift from Gibson T J. Placental DNA is from commercial source (Sigma, UK). SssDNA is sonicated denatured salmon sperm DNA (Sigma, UK). These membranes exist in replicates and are hybridized to different radio-labeled gene probes. 500 ng DNA is loaded per dot.

Table 2a. Grid D correspond to nylon plus membrane that contain DNA from E. coli and gram positive bacteria isolated from patients' fecal specimen. Unless otherwise stated, all are E. coli DNA. Placental DNA, K12 and 0157:H7 are from commercial source. 114/3 g is Streptococcus group D DNA, 115/TA is Streptococcus group G DNA, 116/TC is Aeromonass sobria DNA, 116/TD is Streptococcus viridans DNA, 117/2D is Streptococcus group D DNA, 154/9 is unidentified gram positive bacteria DNA and HP is Helicobacter pylori DNA. 500 ng DNA is loaded per dot.

Table 2a. Grid E correspond to nylon plus membranes that contain in duplicate a range of different amount of Enterobacter cloacae and K12 E. coli DNA. The membranes are then hybridized to different .sup.32P-radiolabeled gene probes t