Application of molecular beacon technology for the identification of bacteria

Scope and method of study. The present study reports the development of a bacterial identification technique based on unique sequences in the 16S rRNA utilizing molecular beacon probes. Molecular beacons are stem loop (hairpin) shaped DNA with a fluorophore and quencher attached to the stem region. In the native hairpin shape of the beacon the fluorophore is quenched by virtue of its proximity to the quencher. Typically, the loop portion of the molecular beacon is complementary to a target sequence. Upon hybridization with the target, the molecular beacon undergoes a conformational shift that moves the fluorophore away from the quencher resulting in fluorescence emission. Molecular beacons targeting unique sequences in the 16S rRNA of three bacterial species, Francisella tularensis, Escherichia coli and Mannheimia haemolytica were developed. The functioning of the F. tularensis molecular beacon was evaluated in solution and immobilized on glass slides, using synthetic targets. Bacterial identification studies were carried out using the E. coli and M. haemolytica molecular beacons.;Findings and conclusions. In solution based experiments molecular beacon functioning was found to be specific, successfully discriminating variable length, single and multiple base mismatch targets from the true target. Hybridization reactions were found to be dependant on changes in MgCl 2 concentration in the buffer. Glass slide-immobilized molecular beacons showed a reduced specificity allowing discrimination of only multiple mismatch targets. The position and number of mismatches in the target was found to affect the specificity of hybridization. It was also shown that after hybridization, treatment of glass slide-immobilized beacons with alkali can render the molecular beacons ready for reuse. Beacons targeting the 16S rRNA of E. coli and M. haemolytica positively identified total RNA samples isolated from the respective bacterial species, but with a low sensitivity. This study paves the way for the development of a standard bacterial-identification assay using molecular beacons, based on unique sequences in the rRNA. Since fluorescence emission from a molecular beacon probe occurs following a hybridization event only, it obviates the need to wash off any unhybridized probes, allowing target detection in a real-time solution-based assay.