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United States Department of Agriculture

Agricultural Research Service

Research Project: IMMUNOLOGY AND INTERVENTION STRATEGIES FOR JOHNE'S DISEASE Title: Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction

Authors
item Leite, Fernando -
item Stokes, Kevin -
item Robbe-Austerman, Suelee -
item Stabel, Judith

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 7, 2012
Publication Date: January 1, 2013
Repository URL: http://handle.nal.usda.gov/10113/56487
Citation: Leite, F.L., Stokes, K.D., Robbe-Austerman, S., Stabel, J.R. 2013. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. Journal of Veterinary Diagnostic Investigation. 25(1):27-34.

Interpretive Summary: Johne's disease is a chronic, debilitating disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle ususally become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms 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 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 whatage 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 ectraction 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: Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal PCR is becoming more widely used today, demonstrating similar sensitivity and specificity to culture. To ensure efficient and reproducible PCR results from a difficult sample matrix such as feces, there are many obstacles that a DNA extraction method must overcome, including the presence of inhibitors and the thick waxy cell wall of MAP. In this study, six commercial DNA extraction kits were evaluated using fecal samples from naturally infected cattle shedding various amounts of MAP. Upon extraction, DNA purity and yield were measured by spectrophotometry, and real-time PCR was performed for detection of the IS900 and ISMAP02 targets. The kits evaluated showed significant differences in the purity and yield of DNA obtained. The best results were observed with Kit E and Kit A, having identified 16/17 (94%) and 13/17 (76%) of the positive samples by IS900 PCR, respectively. This study demonstrates the importance of choosing the correct methodology for the most accurate diagnosis of paratuberculosis through fecal PCR.

Last Modified: 4/21/2014