Rapid and conventional testing methods for microorganisms in milk
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Date
2014Author
Lanyasunya, TP.
Lokwaleput, IK.
Mutunga, TK.
Siamba, DN.
Ondiek, JO.
Bwire, JM.
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mmunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very
effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis
from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp.
paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead
beating; then protein and cellular contaminants were removed by phenol-chloroform–isopropanol extraction
prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional
and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use
of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently
detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and costeffective for the diagnosis of clinical and subclinical Johne’s disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.
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