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 alve~ Enterococcus faecium and Spiroplasma melliferum. The PCR did, however; reliably amplify a 486 bp fragment (when the annealing temperature was lowered by 5°C) using template DNA isolated from the phylagenetically-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 bisbenzidene/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 MP1 and MP2 was developed. The hemi-nested PCR amplified a 276 bp M. plutonspecific product that was not amplified with E faecalis DNA. In sensitivity studies, the PCR assay could reliably detect approximately 1-10 organisms/mi. This level of sensitivity was achieved using crude DNA templates (boiled cell lysate) prepared using lnstagene matrix. The PCR assay could also detect M. pluton in brood with European foulbrood. Downloaded by [University of Birmingham] at 08