Magnetic bead hybridization to detect enterotoxigenicEscherichia colistrains associated with cattle in environmental water sources

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Abstract
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/μL 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.Key words: magnetic bead hybrid, LTIIa toxin gene, water-quality monitoring.

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