It is difficult to detect more than six different targets in a single qPCR assay using current qPCR instruments due to limitations in the fluorophore discriminatory capacity of these instruments. The use of novel 'sloppy' molecular beacon probes in homogeneous qPCR screening assays enables detection of different targets with a much higher degree of multiplexing (as many as 100 targets).
The strategy requires only a single gene amplification assay containing a set of e.g. four differently colored molecular beacon probes of a unique design. After the completion of amplification, the molecular beacons in the reaction tube are hybridized to the amplicons at a relatively low temperature. The temperature is then slowly raised to determine, from the consequent changes in fluorescence intensity of each molecular beacon in the set, the temperature at which each of the four probe-target hybrids melts apart (Tm). The resulting set of four Tm values uniquely identifies the species present in the sample. These assays, despite utilizing only four different probes, have the potential to identify hundred of different bacterial species.
The key to developing single-tube assays that are able to distinguish a long list of species is the selection of a target sequence region that is shared by all of the species of interest, that can be amplified by a pair of universal primers, and that possesses just the right degree of sequence variability so that the presence of each species results in the generation of a unique species-specific signature.
Two different papers describe the application of sloppy molecular beacon probes:
1. Use of Sloppy Molecular Beacon Probes for Identification of Mycobacterial Species. El-Hajj et al. J. Clin. Microbiol. 2009: (47) 1190-1198 Click here for pdf
2. Rapid Universal Identifcation of Bacterial Pathogens from Clinical Cultures by Using a Novel Sloppy Molecular Beacon Melting Temperature Signature Technique. Chakravorty et al., J. Clin. Microbiol. 2010: (48) 258-267 Click here for pdf