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Applied and Environmental Microbiology, August 2006, p. 5150-5158, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00590-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Peptide aMptD-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Bulk Milk Samples

Janin Stratmann, Karen Dohmann, Julia Heinzmann, and Gerald-F. Gerlach*

Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

Received 13 March 2006/ Accepted 8 May 2006

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 102 CFU ml–1 for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 102 CFU ml–1) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 103 and an association constant of 1.33 x 109. The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.

* Corresponding author. Mailing address: Institut für Mikrobiologie, Zentrum für Infektionsmedizin, Stiftung Tierärztliche Hochschule Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany. Phone: 49 511 856 7598. Fax: 49 511 856 7697. E-mail: gfgerlach{at}gmx.de.

Applied and Environmental Microbiology, August 2006, p. 5150-5158, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00590-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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  • Turenne, C. Y., Wallace, R. Jr., Behr, M. A. (2007). Mycobacterium avium in the Postgenomic Era. Clin. Microbiol. Rev. 20: 205-229 [Abstract] [Full Text]  


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