Development of a hemi-nested PCR assay for the specific detection of Melissococcus pluton
Steven P Djordjevic; Kerrie Noone; Lisa Smith; Michael A Z Hornitzky
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°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 HinfI 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/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.
Melissococcus pluton, European foulbrood, polymerase chain reaction, oligonucleotide primers, electrophoresis, Australia