title: Enteroaggregative Escherichia coli quantification in children stool samples using quantitative PCR.
abstract: Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10(5) to 2 × 10(9 ) CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10(7) and 10(6) CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.
authors.affiliation: Institute of Biomedicine for Brazilian Semi-Arid (IBISAB) & Clinical Research Unit, Federal University of Ceara, Fortaleza, Ceara, Brazil.
authors.forename: Ila Fernanda Nunes
authors.initials: IF
authors.lastname: Lima
authors.forename: Josiane da Silva
authors.initials: Jda S
authors.lastname: Quetz
authors.forename: Richard Littleton
authors.initials: RL
authors.lastname: Guerrant
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
authors.forename: Aldo Angelo Moreira
authors.initials: AA
authors.lastname: Lima
authors.forename: Alexandre
authors.initials: A
authors.lastname: Havt
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002648:Child; D004269:DNA, Bacterial / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis*; D029968:Escherichia coli Proteins / Q000235:genetics; D005243:Feces / Q000382:microbiology; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C512825:AatA protein, E coli; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins