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

Abstract

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 Tm 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 Tm values to each primer set, and the melting Tm was measured by hybridizing the stem sequences at both ends of the amplified amplicon; this generated well-separated Tm signals. The important point here is that the stem sequence that produces the Tm signal is an arbitrarily selectable sequence unrelated to the target gene. Because it is arbitrarily selectable, the desired Tm 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.