Development of a hemi-nested PCR assay for the specific detection of Melissococcus pluton

A pair of oligonucleotide primers (MP1 and MP2) were used for the polymerase chain reaction (PCR) amplification of a 486 base pair (bp) fragment of the 16S rRNA gene of 26 geographically diverse Australian Melissococcus pluton (causative agent of European foulbrood) isolates. PCR primers spanning a region of the 16S rRNA gene from position 893-1377 failed to amplify a product when template DNA from a wide range of pathogenic and saprophytic bacteria were used including Paenibacillus larvae, Paenibacillus alvei, Enterococcus faecium and Spiroplasma melliferum. The PCR did, however, reliably amplify a 486 bp fragment (when the annealing temperature was lowered by 5 degrees C) using template DNA isolated from the phylogenetically-related bacterium Enterococcus faecalis. PCR amplicons generated from E. faecalis and M. pluton were readily distinguished by digestion with the restriction endonuclease Hinfl and electrophoresis in 1.5% agarose or by electrophoresis in 1% agarose containing bis-benzidene/polyethylene glycol. A hemi-nested PCR requiring a combination of primers MP1 and a third primer MP3, which spanned 25 nucleotides from position 1168-1144 and internal to the 486 bp amplicon generated by primers MPI and MP2 was developed. The hemi-nested PCR amplified a 276 bp M. pluton-specific product that was not amplified with E. faecalis DNA. In sensitivity studies, the PCR assay could reliably detect approximately 1-10 organisms/ml. This level of sensitivity was achieved using crude DNA templates (boiled cell lysate) prepared using Instagene matrix. The PCR assay could also detect M. pluton in brood with European foulbrood.