|Leite, F -|
Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: February 4, 2012
Publication Date: February 19, 2012
Citation: Leite, F., Stabel, J.R. 2012. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis. In: Proceedings of the 11th International Colloquium on Paratuberculosis, February 5-10, 2012, Sydney, Australia. p. 82-85. Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. Johne’s disease is difficult to diagnose and therefore to control. Development of accurate and sensitive diagnostic tests is dependent upon understanding how the host animal handles the infection and at what age do signs of infection, such as fecal shedding, become obvious. Fecal PCR is becoming widely used for detection of the causative agent of Johne's disease. The current study compares various methods of DNA extraction from fecal samples. Selecting the optimal method of DNA extraction will enhance sensitivity of PCR and provide more accurate diagnosis of paratuberculosis. This will result in improved control and management of the disease in dairy herds.
Technical Abstract: Fecal culture is considered the gold standard for the diagnosis 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 24 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 16/17 (94%) 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.