Use of Sloppy Molecular Beacon Probes for Identification of

We report here the use of novel “sloppy ” molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (Tm) values that identify which species is present in a sample. Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs. By using four sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, four different Tm values can be determined simultaneously. We tested this technique with 27 different species of mycobacteria and found that each species generates a unique, highly reproducible signature that is unaffected by the initial bacterial DNA concentration. Utilizing this general paradigm, screening assays can be designed for the identification of a wide range of species. A classic approach for determining the identity of a bacterial species is to employ PCR to exponentially amplify a selected segment of a 16S rRNA gene (26), utilizing a pair of “universal primers ” that bind to highly conserved sequences at the ends of the target region, and to then use a sophisticated technique t