The present disclosure provides methods, devices and systems that enable simultaneous multiplexing amplification reaction and real-time detection in a single reaction chamber. An aspect of the present disclosure provides a method for assaying a presence or absence of at least one target nucleic acid molecule. In some embodiments of aspects provided herein, at least one signal is produced upon binding of probes to the target nucleic acid molecule and/or the limiting primer. In some embodiments of aspects provided herein, a reaction mixture comprises a plurality of template nucleic acid molecules and the probes specifically bind to a plurality of target nucleic molecules as amplification products of the plurality of template nucleic acid molecules.