Previous Article | Next Article 
Applied and Environmental Microbiology, March 2003, p. 1504-1510, Vol. 69, No. 3
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.3.1504-1510.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization
Tamás Bakonyi,1* Irmgard Derakhshifar,2 Elvira Grabensteiner,3,4 and Norbert Nowotny4,5,6Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, H-1143 Budapest, Hungary,1 Institute for Apiculture, Agricultural Inspection Service and Research Centre Vienna, Austrian Agency for Health and Food Safety and Federal Office for Food Safety, A-1226 Vienna,2 Clinical Virology Group, Institute of Virology,4 Clinic for Poultry and Pet Birds,3 Institute of Hydrobiology, Ichthyology and Apidology,5 University of Veterinary Medicine, A-1210 Vienna, Austria; and Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates6
Received 27 August 2002/ Accepted 25 November 2002
PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.
* Corresponding author. Mailing address: Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Hungária krt. 23-25, H-1143 Budapest, Hungary. Phone: 36 1 251 9900. Fax: 36 1 251 9260. E-mail: bakonyi{at}novell.vmri.hu.
Applied and Environmental Microbiology, March 2003, p. 1504-1510, Vol. 69, No. 3
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.3.1504-1510.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»