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Journal of Clinical Microbiology, March 2004, p. 1075-1081, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1075-1081.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid and Sensitive Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Sangeeta Khare,1 Thomas A. Ficht,1 Renato L. Santos,1 Juan Romano,2 Allison R. Ficht,3 Shuping Zhang,1 Irene R. Grant,4 Melissa Libal,5 David Hunter,6 and L. Garry Adams1*

Department of Veterinary Pathobiology, College of Veterinary Medicine,1 Department of Large Animal Medicine and Surgery, Texas A&M University,2 Department of Medical Biochemistry and Genetics, College of Medicine, Texas A&M University Health Science Center,3 Texas Veterinary Medical Diagnostic Laboratory, College Station, Texas,5 Department of Food Science (Food Microbiology), The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom,4 Turner Enterprises Inc., Bozeman, Montana6

Received 26 February 2003/ Returned for modification 16 May 2003/ Accepted 11 November 2003

Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.

* Corresponding author. Mailing address: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467. Phone: (979) 845-5092. Fax: (979) 845-5088. E-mail: gadams{at}cvm.tamu.edu.

Journal of Clinical Microbiology, March 2004, p. 1075-1081, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1075-1081.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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