title: Improved detection of Escherichia coli and coliform bacteria by multiplex PCR.
abstract: BACKGROUND
  The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified.
  
  
  RESULTS
  In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively.
  
  
  CONCLUSIONS
  The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.
authors.affiliation: Área de Genética, Departamento de Bioquímica y Biologia Molecular y Genética, Universidad de Extremadura, Badajoz, Spain. fmolina@unex.es.
authors.forename: Felipe
authors.initials: F
authors.lastname: Molina
authors.affiliation: Área de Genética, Departamento de Bioquímica y Biologia Molecular y Genética, Universidad de Extremadura, Badajoz, Spain. elpzacedo@gmail.com.
authors.forename: Elena
authors.initials: E
authors.lastname: López-Acedo
authors.affiliation: Dairy products, Technological institute of Food and Agriculture, Badajoz, Spain. rafael.tabla@gobex.es.
authors.forename: Rafael
authors.initials: R
authors.lastname: Tabla
authors.affiliation: Dairy products, Technological institute of Food and Agriculture, Badajoz, Spain. isidro.roa@gmail.com.
authors.forename: Isidro
authors.initials: I
authors.lastname: Roa
authors.affiliation: Dairy products, Technological institute of Food and Agriculture, Badajoz, Spain. toni.gomez.qu@gmail.com.
authors.forename: Antonia
authors.initials: A
authors.lastname: Gómez
authors.affiliation: Área de Genética, Departamento de Bioquímica y Biologia Molecular y Genética, Universidad de Extremadura, Badajoz, Spain. jrebollo@unex.es.
authors.forename: José E
authors.initials: JE
authors.lastname: Rebollo
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D003611:Dairy Products / Q000382:microbiology; D004755:Enterobacteriaceae / Q000145:classification / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins