Polymorphic loci that differentiate escherichia coli 0157:H7 from other strains

Abstract

The present invention relates generally to the field of microbiology and food sciences. More particularly, the inventor has discovered several polynucleotide sequences encoding the gnd gene and corresponding 6-phosphogluconate dehydrogenase (6-PGD) proteins from different strains of Escherichia Coli and polymorphic sequences therein. Novel biotechnological tools, diagnostics, and food screening techniques are provided.

What is claimed is:  
     
         1 . An isolated polynucleotide encoding gnd, wherein the polynucleotide comprises one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34.  
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein the polynucleotide comprises at least 9 consecutive bases of one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 and contains a polymorphism described in Table 1.  
     
     
         3 . The isolated polynucleotide of  claim 1 , wherein the polynucleotide encodes a polypeptide deduced from one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34.  
     
     
         4 . The isolated polynucleotide of  claim 1 , wherein the polynucleotide comprises at least 9 bases that hybridize to the nucleotide sequence of one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 or a sequence complementary thereto under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 pH 7.0, 1 mM EDTA at 50° C.; and washing with 1% SDS at 42° C.  
     
     
         5 . A recombinant construct comprising one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34 operably linked to a heterologous promoter.  
     
     
         6 . A vector comprising the isolated DNA of  claim 1 .  
     
     
         7 . A vector comprising the isolated DNA of  claim 2 .  
     
     
         8 . A method of detecting a polymorphism in a gene encoding 6-PGD comprising: 
 obtaining a biological sample containing polynucleotides; and analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1.    
     
     
         9 . The method of  claim 8 , wherein the polymorphism is C653T or G653C.  
     
     
         10 . The method of  claim 8 , wherein the analysis of the biological sample further comprises a DNA amplification step.  
     
     
         11 . A method of identifying a pathogenic or non-pathogenic  E. coli  comprising: 
 obtaining a biological sample containing polynucleotides;    analyzing the biological sample for the presence of a diagnostic polynucleotide having at least one polymorphism described in Table 1; and    identifying the  E. coli  as a pathogenic or non-pathogenic strain based on the presence or absence of at least one polymorphism described in Table 1.    
     
     
         12 . The method of  claim 11 , wherein the polymorphism is C653T or G653C.  
     
     
         13 . The method of  claim 11 , wherein the analysis of the biological sample further comprises a DNA amplification step.  
     
     
         14 . An isolated protein comprising the sequence of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35.  
     
     
         15 . An isolated polypeptide comprising at least 3 consecutive amino acids of one of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35, wherein the polypeptide contains at least one polymorphism that can be deduced from Table 1.  
     
     
         16 . A method of making a 6-PGD protein comprising: 
 obtaining a cDNA comprising one of the sequences of SEQ ID Nos: 22, 16, 18, 24, 26, 20, 42, 28, 30, 40, 32, 36, 38, and 34;    inserting the cDNA in an expression vector such that the cDNA is operably linked to a promoter; and    introducing the expression vector into a host cell whereby the host cell produces the protein encoded by the cDNA.    
     
     
         17 . The method of  claim 16 , further comprising isolating the protein.  
     
     
         18 . A method for constructing a transformed host cell that expresses one of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35 comprising transforming a host cell with a recombinant DNA vector suitable for gene expression.  
     
     
         19 . A cultured cell line comprising the vector of  claim 6 .  
     
     
         20 . A cultured cell line comprising the vector of  claim 7 .  
     
     
         21 . An isolated antibody capable of specifically binding to a protein having one of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1.  
     
     
         22 . An isolated antibody capable of binding to a polypeptide comprising at least 9 consecutive amino acids of the sequences of SEQ ID Nos: 23, 17, 19, 25, 27, 21, 43, 29, 31, 41, 33, 37, 39, and 35, wherein the epitope corresponds to at least one polymorphism that can be deduced from Table 1.  
     
     
         23 . The antibody of  claim 21  or  22 , wherein the antibody is a monoclonal antibody.  
     
     
         24 . A nucleic acid probe for detecting the presence of  E. coli  O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, said isolated nucleic acid molecule hybridizing to DNA of gnd of  E. coli  O157:H7 and not to DNA of gnd of non-H7  E. coli  O157 strains.  
     
     
         25 . A nucleic acid primer for detecting the presence of  E. coli  O157:H7 consisting of an isolated nucleic acid molecule at least 7 nucleotides in length, said isolated nucleic acid molecule priming DNA of gnd of  E. coli  O157:H7 and not DNA of gnd of non-H7  E. coli  O157 strains.  
     
     
         26 . A method for detecting the presence of  E. coli  O157:H7 in a sample comprising the steps of: 
 (a) contacting said sample, under hybridization conditions, with a nucleic acid probe that selectively hybridizes to a nucleic acid sequence from gnd of  E. coli  O157:H7 and not to nucleic acid sequence from gnd of non-H7  E. coli  O157 strains, to form a hybridization complex; and    (b) detecting formation of said hybridization complex as an indication of the presence of  E. coli  O157:H7 in the sample.    
     
     
         27 . A plurality of the nucleic acid probes of  claim 24  on a substrate.  
     
     
         28 . A plurality of the nucleic acid probes of  claim 24  in a microarray on a chip.