Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization.
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.
Bakonyi
Tamás
T
Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Hungária krt. 23-25, H-1143 Budapest, Hungary. bakonyi@novell.vmri.hu
Derakhshifar
Irmgard
I
Grabensteiner
Elvira
E
Nowotny
Norbert
N
D000818:Animals; D001407:Bacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001516:Bees / Q000254:growth & development / Q000382:microbiology*; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D006722:Honey / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial