Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples

Comparison with isolation and biochemical characterization

Tamás Bakonyi, Irmgard Derakhshifar, Elvira Grabensteiner, Norbert Nowotny

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1504-1510
Number of pages7
JournalApplied and Environmental Microbiology
Volume69
Issue number3
DOIs
Publication statusPublished - Mar 1 2003

Fingerprint

Paenibacillus larvae
Honey
honey
assay
larva
Polymerase Chain Reaction
assays
honey and bee products
extraction method
DNA
sampling
Bacillus
American foul brood
Paenibacillus
saprophytes
Bacillus (bacteria)
metalloproteinases
Endopeptidase K
identification method
methodology

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples : Comparison with isolation and biochemical characterization. / Bakonyi, Tamás; Derakhshifar, Irmgard; Grabensteiner, Elvira; Nowotny, Norbert.

In: Applied and Environmental Microbiology, Vol. 69, No. 3, 01.03.2003, p. 1504-1510.

Research output: Contribution to journalArticle

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