Magnetic bead hybridization to detect enterotoxigenic Escherichia coli strains associated with cattle in environmental water sources.
A magnetic capture hybridization - polymerase chain reaction (MCH-PCR) method was used to increase the detection sensitivity of the enterotoxin gene LTIIa, used as a biomarker for waste in environmental samples. The samples were collected from cow lagoons of different farms and from environmental waters. Total DNA was extracted from colonies grown on mTEC medium or directly from environmental samples. The cow-specific Escherichia coli LTIIa gene was used as a DNA marker. A LTIIa-specific oligonucleotide probe was designed to capture the LTIIa marker during the MCH, followed by PCR. Varying levels of humic acid were added to the DNA extracts to evaluate the sensitivity and effectiveness of MCH-PCR. The minimal detection limit of MCH-PCR for the LTIIa gene was 2.5 ag/muL DNA. In the presence of humic acid, MCH-PCR was able to increase the detection sensitivity 10 000-fold over that of conventional PCR. The MCH-PCR could also detect one cell with the LTIIa DNA marker in a 1-L seeded environmental water sample. Results in this study indicate that MCH-PCR is more sensitive than nested PCR in testing environmental samples.
Department of Environmental Health Science and Policy, University of California, Irvine 92697, USA.
Yu-Li
YL
Tsai
Joanne Y
JY
Le
Betty H
BH
Olson
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D002417:Cattle / Q000382:microbiology*; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D005243:Feces / Q000382:microbiology*; D005798:Genes, Bacterial; D006812:Humic Substances; D008280:Magnetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D006812:Humic Substances; C027538:heat-labile enterotoxin, E coli