Molecular detection of the entomopathogenic bacterium Pseudomonas entomophila using PCR.
OBJECTIVE
To develop a specific, fast and simple molecular method useful to detect the entomopathogenic bacterium Pseudomonas entomophila.


RESULTS
The use of bioinformatics tools allowed the identification of unique genes present in P. entomophila genome. Using such genes, we designed primers aiming to detect specifically P. entomophila by PCR. Furthermore, a pair of primers specifically designed to amplify the 16S rRNA gene in Pseudomonas species was used. Primer specificity was checked using environmental pseudomonad and nonpseudomonad species. A 618 -bp fragment was amplified only in Pseudomonas using the 16S rDNA primers. Primers (PSEEN1497) designed to detect P. entomophila amplified a 570 -bp fragment only in P. entomophila. A duplex PCR was developed combining 16S rDNA and PSEEN1497 primers that allowed the detection of P. entomophila present in experimentally infected Drosophila melanogaster.


CONCLUSIONS
We developed a molecular method useful to detect P. entomophila present in bacterial cultures or directly from infected insects.


CONCLUSIONS
To the best of our knowledge, this is the first molecular method aiming to detect P. entomophila in environmental samples. The use of our method will facilitate studies related to ecology and insect host range of this entomopathogenic bacterium.
Papagiannoulis
A
A
Department of Biochemistry & Biotechnology, University of Thessaly, Larissa, Greece.
Mathiopoulos
K D
KD
Mossialos
D
D
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004331:Drosophila melanogaster / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011549:Pseudomonas / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S