title: Slot blot immunoassay as a tool for plasmid-encoded toxin detection in enteroaggregative Escherichia coli culture supernatants.
abstract: Plasmid-encoded toxin (Pet) is a heat-labile enterotoxin encoded in the enteroaggregative Escherichia coli (EAEC) virulence plasmid. Several evidence support the role of this 108-kDa secreted protein in the pathogenesis of EAEC diarrhea. In this study, we standardized a slot blot immunoassay for Pet detection. EAEC culture supernatants were applied onto a polyvinylidene difluoride membrane, and, using rabbit polyclonal Pet antisera, the expression of the toxin by slot blot immunoassay was observed in 9.5% of the isolates studied. In addition, no negative control reacted with Pet antiserum in this assay. This assay is a rapid, specific, reproducible, and low-cost methodology, therefore demonstrating its potential in diagnosing Pet expression. Moreover, we describe for the first time that expression of Pet can be directly detected from EAEC culture supernatants and may be used in clinical laboratorial routine instead of polymerase chain reaction detection of the pet gene, especially in developing countries where the EAEC pathotype has been considered an emerging pathogen.
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 05503-900 São Paulo, Brazil.
authors.forename: Andréa Bernardes
authors.initials: AB
authors.lastname: Vilhena-Costa
authors.forename: Roxane Maria Fontes
authors.initials: RM
authors.lastname: Piazza
authors.forename: Júlia Mitico
authors.initials: JM
authors.lastname: Nara
authors.forename: Luiz Rachid
authors.initials: LR
authors.lastname: Trabulsi
authors.forename: Marina Baquerizo
authors.initials: MB
authors.lastname: Martinez
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D001431:Bacteriological Techniques; D003470:Culture Media / Q000737:chemistry*; D004768:Enterotoxins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases
chemical_list: D001427:Bacterial Toxins; D003470:Culture Media; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli; C530786:Pet protein, E coli; D012697:Serine Endopeptidases