Simultaneous Detection of Multiple Pathogenic Targets with Stem-Tagged Primer Sets

Simultaneous multiple gene detection is indispensable for the detection of various genes in a small sample obtained by an invasive method. A typical detection method is probe-based fluorescence melting curve analysis by means of real-time PCR. It is very limited because, for each target, a probe sequence with at least a different T_m must be designed. To overcome this limitation, we developed a simultaneous multiple gene detection method based on a giant amplicon molecular beacon. PCR was performed by attaching stem sequences with different T_m values to each primer set, and the melting T_m was measured by hybridizing the stem sequences at both ends of the amplified amplicon; this generated well-separated T_m signals. The important point here is that the stem sequence that produces the T_m signal is an arbitrarily selectable sequence unrelated to the target gene. Because it is arbitrarily selectable, the desired T_m can be freely adjusted. As a result, we succeeded in the simultaneous detection of four samples with the use of only one fluorophore. Theoretically, a combination of five fluorophores could detect more than 20 multiple genes simultaneously.