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OJVRTM
Online Journal of Veterinary
Research©
Volume 16 (4):163-171, 2012. Redacted 2017.
Optimization of DNA Extraction for identification of Mycobacterium avium
subsp. paratuberculosis
in faeces
1Faculty of Veterinary Medicine, Benha
University, P.O. Box 13736, Mostohor, Toukh, Egypt. 2State
Institute for Consumer Protection of Saxony-Anhalt, Dept. of Veterinary
Medicine, P.O. Box D-39576, Haferbreiter Weg Str.132-135, Stendal, Germany
SUMMARY
Selim A, Gaede W., Optimization of DNA Extraction for identification of
Optimization of DNA Extraction for identification of Mycobacterium avium subsp. paratuberculosis in faeces, Onl J Vet Res., 16 (4):163-171, 2012. Diagnosis of Johne`s disease, an enteric infection of cattle caused by Mycobacterium avium
subsp. paratuberculosis
requires a rapid, cheap, sensitive and reliable test for detection. A method to
estimate and evaluate extraction MAP-DNA from field faeces
from contaminated faecal samples of cattle using
IS900 and ISMAV2 real-time PCR with internal control (PUC19 plasmid) is
described. The analysis of MAP-spiked faecal samples
resulted in a reproducible detection limit of 100 MAP-cells/g faeces for IS900-PCR and ISMAV2-PCR if either the modified High
Pure PCR Template Preparation kit® or the magnetic beads method had been used
for DNA-isolation. The application of Adiapure® Purification and Extraction kit
enabled the detection of 100 MAP-cells/g faeces when followed by IS900-PCR,
otherwise reproducible detection limit increased to 1000 MAP-cells/g faeces if
the Adiapure®-templates were analysed with ISMAV2-PCR. The results for spiked
faecal samples indicated that the most sensitive detection of MAP in bovine
faeces can be assued by the combination of IS900-PCR or ISMAV2-PCR with the
modified application of the High Pure PCR Template Preparation kit®.
Key words: MAP, feces, IS900-PCR, ISMAV2-PCR
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