|Leite, Fernando -|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 11, 2011
Publication Date: February 4, 2012
Citation: Leite, F., Stabel, J.R. 2012. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis [abstract]. 11th International Colloquium on Paratuberculosis. p. 196. Technical Abstract: Fecal culture is considered the gold standard for the diagnostics of paratuberculosis, however, PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal material is widely used today, having demonstrated great sensitivity and specificity. To insure the most efficient and reproducible PCR assay, there are many obstacles that a DNA extraction method needs to overcome, including the presence of inhibitors in feces and the thick waxy cell wall of MAP. In this study, we compared six commercial fecal DNA extraction kits for their ability to extract DNA from fecal samples of animals shedding MAP. Samples obtained from 25 animals shedding different levels of bacteria as characterized by fecal culture were extracted blindly in duplicate. Real-time PCR was done for the insertion sequences IS900 and ISMap02, and DNA purity and yield were measured by spectrophotometry. The kits evaluated were: MagMax™ Total Nucleic Acid Isolation Kit (Applied Biosystems™), PowerSoil® DNA Isolation Kit (MO BIO Laboratories), ZR Fecal DNA MiniPrep™ (Zymo Research), ExtractMaster™ Fecal DNA Extraction Kit (Epicenter® Biotechnologies), Tetracore® MAP Extraction System (Tetracore®) and QIAamp® Stool DNA Mini Kit (Quiagen). The kits evaluated showed significant differences amongst each other in the purity and yield of DNA obtained, as well as different sensitivities in identifying MAP DNA in animals shedding the bacteria. All of the kits had good reproducibility between the duplicate samples. The best results were observed with the ZR Fecal DNA MiniPrep kit and the MagMax™ Total Nucleic Acid Isolation Kit, having identified 15/17 (88%) and 13/17 (76%) of the positive samples, respectively. This study demonstrates the importance of choosing the correct methodology for the most accurate diagnosis of paratuberculosis through fecal PCR.