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Applied and Environmental Microbiology, May 2008, p. 2751-2758, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02534-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

New Triplex Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Feces{triangledown}

H. Schönenbrücher,1* A. Abdulmawjood,1 K. Failing,2 and M. Bülte1

Institute of Veterinary Food Science, Faculty of Veterinary Medicine, Justus Liebig University of Giessen, Frankfurter Str. 92, D-35392 Giessen, Germany,1 Unit for Biomathematics and Data Processing, Faculty of Veterinary Medicine, Justus Liebig University of Giessen, Frankfurter Str. 95, D-35392 Giessen, Germany2

Received 9 November 2007/ Accepted 24 February 2008

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.

* Corresponding author. Mailing address: Institute of Veterinary Food Science, Faculty of Veterinary Medicine, Justus Liebig University of Giessen, Frankfurter Str. 92, D-35392 Giessen, Germany. Phone: 49 641-99-38251. Fax: 49 641-99-38259. E-mail: Holger.Schoenenbruecher{at}vetmed.uni-giessen.de

{triangledown} Published ahead of print on 7 March 2008.

Applied and Environmental Microbiology, May 2008, p. 2751-2758, Vol. 74, No. 9
0099-2240/08/$08.00+0     doi:10.1128/AEM.02534-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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