title: Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool.
abstract: Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.
authors.affiliation: Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA. mt2f@virginia.edu
authors.forename: Mami
authors.initials: M
authors.lastname: Taniuchi
authors.forename: Carol C
authors.initials: CC
authors.lastname: Walters
authors.forename: Jean
authors.initials: J
authors.lastname: Gratz
authors.forename: Athanasia
authors.initials: A
authors.lastname: Maro
authors.forename: Happiness
authors.initials: H
authors.lastname: Kumburu
authors.forename: Oralak
authors.initials: O
authors.lastname: Serichantalergs
authors.forename: Orntipa
authors.initials: O
authors.lastname: Sethabutr
authors.forename: Ladaporn
authors.initials: L
authors.lastname: Bodhidatta
authors.forename: Gibson
authors.initials: G
authors.lastname: Kibiki
authors.forename: Denise M
authors.initials: DM
authors.lastname: Toney
authors.forename: Lynette
authors.initials: L
authors.lastname: Berkeley
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
mesh_terms: D015169:Colony Count, Microbial / Q000379:methods; D003967:Diarrhea / Q000382:microbiology*; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D006801:Humans; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012760:Shigella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D018709:Statistics, Nonparametric