Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR

Y. HU, Q. ZHANG and J.C. MEITZLER.1999.Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt‐I, slt‐II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non‐O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga‐like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross‐react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.

[1]  B. Bosworth,et al.  Multiplex PCR for Enterotoxigenic, Attaching and Effacing, and Shiga Toxin-Producing Escherichia coli Strains from Calves , 1998, Journal of Clinical Microbiology.

[2]  P. Desmarchelier,et al.  A PCR Specific for Escherichia coli O157 Based on the rfb Locus Encoding O157 Lipopolysaccharide , 1998, Journal of Clinical Microbiology.

[3]  I. Nagano,et al.  Detection of Verotoxin‐Producing Escherichia coli O157:H7 by Multiplex Polymerase Chain Reaction , 1998, Microbiology and immunology.

[4]  S. Altekruse,et al.  FOODBORNE DISEASES: Emerging Pathogens and Trends * , 1998 .

[5]  James C. Paton,et al.  Detection and Characterization of Shiga ToxigenicEscherichia coli by Using Multiplex PCR Assays forstx1, stx2,eaeA, Enterohemorrhagic E. coli hlyA,rfbO111, andrfbO157 , 1998, Journal of Clinical Microbiology.

[6]  C. Gerba,et al.  PCR Amplification and Species Determination of Microsporidia in Formalin-Fixed Feces after Immunomagnetic Separation , 1998, Applied and Environmental Microbiology.

[7]  M. Saarela,et al.  Detection of Porphyromonas gingivalisfrom Saliva by PCR by Using a Simple Sample-Processing Method , 1998, Journal of Clinical Microbiology.

[8]  T. Cebula,et al.  Detection and characterization of thefimAgene ofEscherichia coliO157:H7 , 1997 .

[9]  S. Goyal,et al.  Comparison of four RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus by RT-PCR. , 1997, The new microbiologica.

[10]  E Isogai,et al.  Direct Detection by PCR of Escherichia coli O157 and Enteropathogens in Patients with Bloody Diarrhea , 1997, Microbiology and immunology.

[11]  K. Venkateswaran,et al.  A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR , 1997, Applied and environmental microbiology.

[12]  S. Altekruse,et al.  Emerging foodborne diseases. , 1997, Emerging infectious diseases.

[13]  R. Speich,et al.  Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens , 1997, Journal of clinical microbiology.

[14]  E. Galun,et al.  Comparison of methods for extraction of nucleic acid from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences , 1997, Journal of clinical microbiology.

[15]  R. Lindqvist.  Preparation of PCR samples from food by a rapid and simple centrifugation technique evaluated by detection of Escherichia coli O157:H7. , 1997, International journal of food microbiology.

[16]  P. Feng.  Impact of molecular biology on the detection of foodborne pathogens , 1997, Molecular biotechnology.

[17]  M. Amills,et al.  Isolation of genomic DNA from milk samples by using Chelex resin , 1997, Journal of Dairy Research.

[18]  R. Lindqvist,et al.  A rapid sample preparation method for PCR detection of food pathogens based on buoyant density centrifugation , 1997, Letters in applied microbiology.

[19]  F. Mégraud,et al.  Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model , 1997, Journal of clinical microbiology.

[20]  V. Gannon,et al.  Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains , 1997, Journal of clinical microbiology.

[21]  S. Kresovich,et al.  A multiplex PCR for identifying Shiga‐like toxin‐producing Escherichia coli O157 : H7 , 1997, Letters in applied microbiology.

[22]  P. Choudary,et al.  Rapid and sensitive immunomagnetic separation–polymerase chain reaction method for the detection of Escherichia coli O157[ratio ]H7 in raw milk and ice-cream , 1997, Journal of Dairy Research.

[23]  M. Wolf,et al.  Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine , 1997, Infection and immunity.

[24]  T. Cebula,et al.  Detection and characterization of the fimA gene of Escherichia coli O157:H7. , 1997, Molecular and cellular probes.

[25]  W. L. Payne,et al.  High Mutation Frequencies Among Escherichia coli and Salmonella Pathogens , 1996, Science.

[26]  S. Dowell,et al.  Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus , 1996, Infection and immunity.

[27]  S. Kresovich,et al.  Polymerase chain reaction for detecting Escherichia coli O157: H7. , 1996, International journal of food microbiology.

[28]  P. Feng,et al.  Emergence of rapid methods for identifying microbial pathogens in foods. , 1996, Journal of AOAC International.

[29]  K. Livak,et al.  A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef , 1996, Applied and environmental microbiology.

[30]  M. Leinonen,et al.  PCR assay for detecting Streptococcus pneumoniae in the middle ear of children with otitis media with effusion. , 1996, Acta oto-laryngologica.

[31]  L. Fox,et al.  Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces , 1995, Journal of clinical microbiology.

[32]  C. Dorn,et al.  DNA probe for detection of serogroup O157 enterohemorrhagic Escherichia coli. , 1995, International journal of food microbiology.

[33]  T. McDaniel,et al.  A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[34]  W. L. Payne,et al.  Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR , 1995, Journal of clinical microbiology.

[35]  P. Feng,et al.  Genetic analysis of uidA expression in enterohaemorrhagic Escherichia coli serotype O157:H7. , 1994, Microbiology.

[36]  T. Cheasty,et al.  Hybridization of strains of Escherichia coli O157 with probes derived from the eaeA gene of enteropathogenic E. coli and the eaeA homolog from a Vero cytotoxin-producing strain of E. coli O157 , 1994, Journal of clinical microbiology.

[37]  B. Swaminathan,et al.  Rapid detection of food-borne pathogenic bacteria. , 1994, Annual review of microbiology.

[38]  P. Fratamico,et al.  Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant , 1993, Applied and environmental microbiology.

[39]  P. Manning,et al.  Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction , 1993, Journal of clinical microbiology.

[40]  J. Thirlwell,et al.  Examination of raw beef products for the presence of Vero cytotoxin producing Escherichia coli, particularly those of serogroup O157. , 1993, The Journal of applied bacteriology.

[41]  D. Francis,et al.  Characteristics of vero cytotoxin producing Escherichia coli associated with intestinal colonization and diarrhea in calves , 1993, Veterinary Microbiology.

[42]  V. Gannon,et al.  Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction , 1993, Journal of clinical microbiology.

[43]  N. Strockbine,et al.  Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen , 1993, Journal of clinical microbiology.

[44]  H. Smith,et al.  ACP Broadsheet 135: January 1993. Isolation and identification methods for Escherichia coli O157 and other Vero cytotoxin producing strains. , 1993, Journal of clinical pathology.

[45]  V. Gannon,et al.  Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction , 1992, Applied and environmental microbiology.

[46]  J. Heesemann,et al.  Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation , 1992, Infection and immunity.

[47]  J. Yu,et al.  Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7 , 1992, Molecular microbiology.

[48]  Ø. Olsvik,et al.  A nested PCR followed by magnetic separation of amplified fragments for detection of Escherichia coli Shiga-like toxin genes. , 1991, Molecular and cellular probes.

[49]  C. Dorn,et al.  Serotype O157:H7 Escherichia coli from bovine and meat sources , 1991, Journal of clinical microbiology.

[50]  D. Milford,et al.  Literature abstracts , 1991, The Lancet.

[51]  B. Rowe,et al.  Properties of Vero cytotoxin-producing Escherichia coli of human and animal origin belonging to serotypes other than O157: H7 , 1989, Epidemiology and Infection.

[52]  S. Ratnam,et al.  Latex agglutination test for detection of Escherichia coli serotype O157 , 1989, Journal of clinical microbiology.

[53]  S. Ratnam,et al.  Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis , 1986, Journal of clinical microbiology.