TaqManTM-PCR for the detection of pathogenic E.coli strains

Abstract

The present invention relates to a method for the detection of pathogenic E. coli in a sample comprising PCR amplification of DNA isolated from said sample using oligonucleotide primers specific for pathogenic E. coli.

1 . A method for the detection of pathogenic  E. coli  in a sample comprising PCR amplification of DNA isolated from said sample using a set of oligonucleotide primers specific for virulence factors/toxins of pathogenic  E. coli  selected from 
 primers that hybridise to a gene encoding heat labile toxin, or heat stabile toxin for the amplification of a DNA sequence characteristic for enterotoxigenic  E. coli;      primers that hybridise to a gene encoding heat stabile toxin for the amplification of a DNA sequence characteristic for enteroaggregative  E. coli;      primers that hybridise to the pCVD432 plasmid for the amplification of a DNA sequence characteristic for enteroaggregative  E. coli;      primers that hybridise to the inv-plasmid for the amplification of a DNA sequence contained in enteroinvasive  E. coli;      primers that hybridise to the EAF plasmid, or the eae gene for the amplification of a DNA sequence characteristic for enteropathogenic  E. coli ; and/or    primers that hybridise to the genes encoding shiga-like toxin sltI or sltII for the amplification of a DNA sequence characteristic for enterohemorrhagic  E. coli , followed by detection and identification of the amplified product using conventional methods.    
     
     
         2 . The method according to  claim 1  wherein 
 the set of primers that hybridise to the gene encoding heat labile toxin characteristic for enterotoxigenic  E. coli  is  
                                       LT-1:   5′ GCG TTA CTA TCC TCT CTA TGT G 3′             and                   LT-2:   5′ AGT TTT CCA TAC TGA TTG CCG C 3′;                              
 the set of primers that hybridise to the gene encoding heat stabile toxin characteristic for enterotoxigenic  E. coli  is  
                                       ST-1:   5′ TCC CTC AGG ATG CTA AAC CAG 3′             and                   ST-2a:   5′ TCG ATT TAT TCA ACA AAG CAA C 3′;                              
 the set of primers that hybridise for the gene encoding heat stabile toxin characteristic for enteroaggregative  E. coli  is  
                                       EASTI-1:   5′ AAC TGC TGG GTA TGT GGC TGG 3′             and                   EASTI-2:   5′ TGC TGA CCT GCC TCT TCC ATG 3′;                              
 the set of primers which hybridise to the pCVD432 plasmid is  
                                       EA-1:   5′ CTG GCG AAA GAC TGT ATC ATT G 3′             and                   EA-2:   5′ TAA TGT ATA GAA ATC CGC TGT T 3′;                              
 the set of primers which hybridise to the inv-plasmid is  
                                       EI-1:   5′ TTT CTG GAT GGT ATG GTG AGG 3′             and                   EI-2:   5′ CTT GAA CAT AAG GAA ATA AAC 3′;                              
 the set of primers which hybridise to the EAF plasmid is  
                                       EP-1:   5′ CAG GGT AAA AGA AAG ATG ATA AG 3′             and                   EP-2:   5′ AAT ATG GGG ACC ATG TAT TAT C 3′;                              
 the set of primers which hybridise to the eae gene is  
                                       EPeh-1:   5′ CCC GGA CCC GGC ACA AGC ATA AG 3′             and                   EPeh-2:   5′ AGT CTC GCC AGT ATT CGC CAC C 3′;                              
 the primers which hybridises to the gene encoding shiga-like toxin SltI is  
                           SltI-1:   5′ ATG AAA AAA ACA TTA TTA ATA GC 3′         and           SltI-2:   5′ TCA CYG AGC TAT TCT GAG TCA AGC 3′;     and                           
 the primers which hybridises to the gene encoding shiga-like toxin SltII is  
                       SltII-1:         5′ ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G 3′     and           SltII-2:     5′ TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC 3′                            
 wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K is G/T.  
 
     
     
         3 . The method according to  claims 1  to  2  wherein a polymerase having additional 5′-3′ exonuclease activity is used for the amplification of DNA, and an oligonucleotide probe labelled at the most 5′ base with a fluorescent dye and at the most 3′ base with a fluorescent quencher dye which hybridises within the target DNA is included in the amplification process; said labelled oligonucleotide probe being susceptible to 5′-3′ exonuclease degradation by said polymerase to produce fragments that can be detected by fluorogenic detection methods.  
     
     
         4 . The method according to  claim 3  wherein 
 the labelled oligonucleotide probe for the detection of heat labile toxin characteristic for enterotoxigenic  E. coli  is  
   5′  AGC TCC CCA CTC TAT TAC AGA ACT ATG  3′ ; 
 the labelled oligonucleotide probe for the detection of heat stabile toxin characteristic for enterotoxigenic  E. coli  is  
   5′  ACA TAC GTT ACA GAC ATA ATC AGA ATC AG  3′ ; 
 the labelled oligonucleotide probe for the detection of heat stabile toxin characteristic for enteroaggregative  E. coli  is  
   5′  ATG AAG GGG CGA AGT TCT GGC TCA ATG TGC  3′ ; 
 the labelled oligonucleotide probe for the detection of pCVD432 plasmid is  
   5′  CTC TTT TAA CTT ATG ATA TGT AAT GTC TGG  3′ ;  
 the labelled oligonucleotide probe for the detection of the inv-plasmid is;  
   5′  CAA AAA CAG AAG AAC CTA TGT CTA CCT  3′ ; 
 the labelled oligonucleotide probe for the detection of the EAF-plasmid is;  
 5′° CTT GGA GTG ATC GAA CGG GAT CCA AAT  3′ ; 
 the labelled oligonucleotide probe for the detection of the eae gene is  
   5′  TAA ACG GGT ATT ATC AAC AGA AAA ATC C  3′ ; 
 the labelled oligonucleotide probe for the detection of shiga-like toxin SltI gene is  
   5′  TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA  3′ ; and 
 the labelled oligonucleotide probe for the detection of shiga-like toxin SltII gene is  
   5′  CAG GTA CTG CAT TTG ATT GTG ACA GTC ATT  3′ . 
 
     
     
         5 . The method according to  claims 3  to  4  wherein the fluorescent reporter dye is 6-carboxy-fluoroscein, tetrachloro-6-carboxy-fluoroscein, or hexachloro-6-carboxy-fluoroscein, and the fluorescent quencher dye is 6-carboxytetramethyl-rhodamine.  
     
     
         6 . The method according to  claims 1  to  5  wherein the PCR amplification process consists of 35 PCR cycles at a MgCl 2  concentration of 5.2 mmol, an annealing temperature of 55° C. and an extension temperature of 65° C.  
     
     
         7 . A set of primers useful for PCR amplification of DNA specific for virulence factors/toxins of pathogenic  E. coli  selected from: 
 a set of primers that hybridise to a gene encoding heat labile toxin, or heat stabile toxin of enterotoxigenic  E. coli;      a set of primers that hybridise to a gene encoding heat stabile toxin of enteroaggregative  E. coli;      a set of primers that hybridise to the pCVD432 plasmid of enteroaggregative  E. coli;      a set of primers that hybridise to the inv-plasmid of enteroinvasive  E. coli;      a set of primers that hybridise to the EAF plasmid, or the eae gene of enteropathogenic  E. coli;  and    a set of primers that hybridise to the gene encoding shiga-like toxin sltl or sltII of enterohemorrhagic  E. coli,      
     
     
         8 . The set of primers according to  claim 7  wherein 
 the set of primers which hybridise to the gene encoding heat labile toxin of enterotoxigenic  E. coli  is  
                                       LT-1:   5′ GCG TTA CTA TCC TCT CTA TGT G 3             and                   LT-2:   5′ AGT TTT CCA TAC TGA TTG CCG C 3′;                              
 the set of primers which hybridise to the gene encoding heat stabile toxin of enterotoxigenic  E. coli  is  
                                       ST-1:   5′ TCC CTC AGG ATG CTA AAC CAG 3′             and                   ST-2a:   5′ TCG ATT TAT TCA ACA AAG CAA C 3′;                              
 the set of primers which hybridise to the gene encoding heat stabile toxin of enteroaggregative  E. coli  is  
                                       EASTI-1:   5′ AAC TGC TGG GTA TGT GGC TGG 3′             and                   EASTI-2:   5′ TGC TGA CCT GCC TCT TCC ATG 3′;                              
 the set of primers which hybridise to the pCVD432 plasmid is  
                                       EA-1:   5′ CTG GCG AAA GAC TGT ATC ATT G 3′             and                   EA-2:   5′ TAA TGT ATA GAA ATC CGC TGT T 3′;                              
 the set of primers which hybridise to the inv-plasmid is  
                                       EI-1:   5′ TTT CTG GAT GGT ATG GTG AGG 3′             and                   EI-2:   5′ CTT GAA CAT AAG GAA ATA AAC 3′;                              
 the set of primers which hybridise to the EAF plasmid is  
                                       EP-1:   5′ CAG GGT AAA AGA AAG ATG ATA AG 3′             and                   EP-2:   5′ AAT ATG GGG ACC ATG TAT TAT 3′;                              
 the set of primers which hybridise to the eae gene is  
                                       EPeh-1:   5′ CCC GGA CCC GGC ACA AGC ATA AG 3′             and                   EPeh-2:   5′ AGT CTC GCC AGT ATT CGC CAC C 3′;                              
 the set of primers which hybridise to the shiga-like toxin sltI gene is  
                           SltI-1:   5′ ATG AAA AAA ACA TTA TTA ATA GC 3′         and           SltI-2:   5′ TCA CYG AGC TAT TCT GAG TCA AGC 3′;                          
 and  
 the set of primers which hybridise to the shiga-like toxin sltII is  
                       SltII-1:         5′ ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G 3′     and           SltII-2:     5′ TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC 3′                            
 wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K is G/T.  
 
     
     
         9 . The set of primers according to  claim 8  which in addition to the primers for amplification of target DNA comprise a labelled oligonucleotide probe which is labelled with a fluoroscent reporter dye, such as 6-carboxy-fluoroscein, tetrachloro-6-carboxy-fluoroscein, hexachloro-6-carboxy-fluoroscein, at the most 5′ base and a fluoroscent quencher dye, such as 6-carboxytetramethyl-rhodamine, at the most 3′ base, and have a nucleotide sequence selected from  
         5′  AGC TCC CCA GTC TAT TAC AGA ACT ATG  3′   
       which hybridises to a gene encoding heat labile toxin of enterotoxigenic  E. coli;    
         5′  ACA TAC GTT ACA GAC ATA ATC AGA ATC AG  3′   
       which hybridises to a gene encoding heat stabile toxin of enterotoxigenic  E. coli;    
         5′  ATG AAG GGG CGA AGT TCT GGC TCA ATG TGC  3′   
       which hybridises to a gene encoding heat stabile toxin of enteroaggregative  E. coli;    
         5′  CTC TTT TAA CTT ATG ATA TGT AAT GTC TGG  3′   
       which hybridises to the pCVD432 plasmid;  
         5′  CAA AAA CAG AAG AAC CTA TGT CTA CCT  3′   
       which hybridises to the inv-plasmid;  
         5′  CTT GGA GTG ATC GAA CGG GAT CCA AAT  3′   
       which hybridises to the EAF plasmid;  
         5′  TAA ACG GGT ATT ATC AAC AGA AAA ATCC  3′   
       which hybridises to the eae gene;  
         5′  TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA  3′   
       which hybridises to the shiga-like toxin SltI gene; and  
         5′  CAG GTA CTG GAT TTG ATT GTG ACA GTC ATT  3′   
       which hybridises to the shiga-like toxin SltII gene.  
     
     
         10 . The use of the method according to  claims 1  to  6  for diagnosing an  E. coli  infection of a living animal body, including a human, or for the detection of  E. coli  contamination of consumables, such as meat, milk and vegetables.