Bacillus. oleronius is a non-motile Gram-negative endospore forming bacterium, isolated for the first time in 1995 from the hindgut of termites. This bacterium has been reported to be the likely cause of demodecosis in humans and therefore it is considered to be a pathogen. The aim of this study was to design novel species - specific primers for B. oleronius and to prove that the primers designed by Szkaradkiewicz et al., 2012 are not specific to B. oleronius as
claimed by the authors.
In order to design-species specific primers, the first objective was to obtain sequence information. Because the bacterium could be cultures easily, the hope was obtain sufficient genomic DNA for single molecule real time sequencing. Extraction the DNA of B. oleronius was challenging. Two extraction kits and three protocols were used to detect which would be the best purification methods for obtaining large amounts of high-quality genomic DNA. The DNA extracts obtained with three different DNA extraction protocols (Gentra Puregene Yeast/Bact kit Gram-positive protocol; Gentra Puregene Yeast/Bact kit Gram-negative protocol; DNeasy ® Blood & Tissue) were measured
by spectrophotometry. DNA extract quantity and purity was varied betwee
n the different extraction kits and A260/A280 and A260/A230 rat ios that used to assess the purity of DNA and nucleic acid respectively indicated
presence of contamination during the extraction procedure. Based on the
obtained results, all the purification methods used in this study failed to
produce pure and high-quality genomic DNA for sequencing. The next objective was to design universal primers for four vary fast evolving genes, single copy gene s (rpoB, recA, gyrB and ytcP) and the ITS region of Bacillus
species for Sanger sequencing and to design species-specific16S RNA primers. To do this, the genetically closest Bacillus species to B. oleronius were identified first through aphylogenetic analysis.
Through this study, it was possible to design two species-specific primer sets for B. oleronius and prove that the primers published by Szkaradkiewicz et al., 2012 are not specific. The new designed primer sets with the publisher’s primers were tested using PCR for B. oleronius as positive control and other bacterial species B. amyloliquefaciens, B. pumilus, B. subtilis, B. licheniformis,
B. sporothermodurans, B. aquimaris, B. carboniphalius and Amphibacillus. tropicus as negative controls. The results showed that the new primer sets
bound specifically to B. oleronius while the publisher’s primers amplified non-specifically fragments of varying sizes of all B acillus species. Based on the results, there is no evidence that B. oleronius was positively identified as being
associated with demodecosis as claimed by Szkaradkiewicz et al., 2012