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Molecular Detection of Verotoxigenic Escherichia coli (VTEC) from Animal Feces for Screening VTEC-shedders |
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Y. Kobayashi, M. Sato, H. Taguchi, S. Koike, H. Nakatsuji, K. Tanaka |
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| Abstract |
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Seventy-six animals including cattle, sheep, horses, 6 species of zoo animals were employed for collection of fresh feces in order to detect verotoxigenic Esherichia coli (VTEC) by safe, quick and sensitive PCR-based molecular methods. Bacterial cell disruption with bead-beating followed by bacterial DNA purification with hydroxyapatide chromatography and gel filtration allowed DNA preparation from animal feces with high recovery and purity. A mountain goat was firstly shown by PCR and sequencing to shed verotoxin 2 gene (vt2) that was used to generate vt2 probe and second primer set for nested PCR to attempt more sensitive detection. Most sensitive nested PCR revealed that 45% of tested cattle and 47% of tested zoo animals were VTEC-positive, while least sensitive normal PCR detected VTEC from none of these animals except a mountain goat. Moderately sensitive detection by PCR in combination with hybridization suggested that the VTEC density varied between the VTEC-positive cattle |
| Keywords:
Verotoxin; E. coli; Cattle; Mountain Goat; PCR; Hybridization |
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