title: Real-Time TaqMan PCR Assay for the Detection of Heat-Labile and Heat-Stable Enterotoxin Genes in a Geographically Diverse Collection of Enterotoxigenic Escherichia coli Strains and Stool Specimens.
abstract: Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of 5 years in developing countries and are the leading bacterial agent of traveler's diarrhea in persons traveling to these countries. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by causing water and electrolyte imbalance. We describe the validation of a real-time TaqMan PCR (RT-PCR) assay to detect LT, ST1a, and ST1b enterotoxin genes in E. coli strains and in stool specimens. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay using a conventional PCR assay as a gold standard with 188 ETEC strains and 42 non-ETEC strains. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay in stool specimens (n = 106) using traditional culture as the gold standard. RT- PCR assay sensitivities for LT, ST1a, and ST1b detection in strains were 100%, 100%, and 98%; specificities were 95%, 98%, and 99%, and Pearson correlation coefficient r was 0.9954 between RT-PCR assay and the gold standard. In stool specimens, RT-PCR assay sensitivities for LT, ST1a, and ST1b detection were 97%, 100%, and 97%; and specificities were 99%, 94%, and 97%. Pearson correlation coefficient r was 0.9975 between RT-PCR results in stool specimens and the gold standard. Limits of detection of LT, ST1a, and ST1b by RT-PCR assay were 0.1 to1.0 pg/μL and by conventional PCR assay were 100 to1000 pg/μL. The accuracy, rapidity and sensitivity of this RT-PCR assay is promising for ETEC detection in public health/clinical laboratories and for laboratories in need of an independent method to confirm results of other culture independent diagnostic tests.
authors: National Center for Zoonotic and Emerging Infectious Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia .
authors: Vaishnavi
authors: V
authors: Pattabiraman
authors: National Center for Zoonotic and Emerging Infectious Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia .
authors: Michele B
authors: MB
authors: Parsons
authors: National Center for Zoonotic and Emerging Infectious Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia .
authors: Cheryl A
authors: CA
authors: Bopp
mesh_terms: D002487:Centers for Disease Control and Prevention, U.S.; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004196:Disease Outbreaks; D004403:Dysentery / Q000382:microbiology*; D054307:Enterotoxigenic Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D062665:Epidemiological Monitoring; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D006358:Hot Temperature; D006801:Humans; D057230:Limit of Detection; D058889:Molecular Typing; D060885:Multiplex Polymerase Chain Reaction; D055550:Protein Stability; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D014481:United States / Q000453:epidemiology
chemical_list: D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins