Species-specific PCR as a tool for the identification of Burkholderia gladioli

Burkholderia gladioli colonizes the respiratory tracts of patients with cys tic fibrosis and chronic granulomatous disease. However, due to the high de gree of phenotypic similarity between this species and closely related spec ies in the Burkholderia cepacia complex, accurate identification is difficu lt, Incorrect identification of these species may have serious repercussion s for the management of patients with cystic fibrosis, To develop an accura te procedure for the identification of B. gladioli, a molecular method to d iscriminate between this species and other species commonly isolated from t he sputa of patients dth cystic fibrosis was investigated. The 23S ribosoma l DNA was cloned from several clinical isolates of B. gladioli, and the nuc leotide sequence was determined. Computer-assisted sequence comparisons ind icated four regions of the 23S rRNA specific for this species; these region s were used to design three primer pairs for species-specific PCR Two of th e printer pairs showed 100% sensitivity and specificity for B. gladioli whe n tested against a panel of 47 isolates comprising 19 B, gladioli isolates and 28 isolates of 16 other bacterial species, One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates o btained from the Burkholderia cepacia Research Laboratory and Repository. T he species-specific PCR nas positive for 70 of 73 isolates of B, gladioli a nd was negative for all other bacterial species examined. Overall, this pri mer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100% , respectively, These data demonstrate the potential of species-specific PC R for the identification of B. gladioli.