Journal article
Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR
Journal of microbiological methods, Vol.99(1), pp.58-65
04/2014
Handle:
https://hdl.handle.net/2376/112054
PMID: 24534783
Abstract
Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease in ruminants and possibly associated with human Crohn's disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (p<0.05). When the pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen), PCR inhibition was not detected in any of three different RT-PCR methods tested in this study. The results obtained with the IS900 probe showed an excellent Kappa value (0.849) and a high correlation coefficient r (0.940) compared to the results of culture method. When used to examine unknown field samples (n=15), more positive tissues were identified with DNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification.
•Three DNA extraction methods of M. paratuberculosis (Map) were evaluated.•All three methods showed different levels of real-time PCR inhibitions.•We present a novel pre-treatment method for Map DNA extraction from tissues.•The method significantly removed PCR inhibitors and increased Map DNA recovery.•The method increased the sensitivity and accuracy of real-time PCR.
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Details
- Title
- Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR
- Creators
- Kun Taek Park - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, United StatesAndrew J Allen - Department of Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, United StatesWilliam C Davis - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, United States
- Publication Details
- Journal of microbiological methods, Vol.99(1), pp.58-65
- Academic Unit
- Veterinary Clinical Sciences, Department of; Veterinary Microbiology and Pathology, Department of
- Publisher
- Elsevier B.V
- Identifiers
- 99900547707901842
- Language
- English
- Resource Type
- Journal article