Development of multiplex PCR for fast detection of Paenibacillus Larvae in putrid masses and in isolated bacterial colonies

The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies?? DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies.