FIG. 1.
Relative orientations of amplification primers, anchors, and detection probes targeted to three different mutation sites of the β-lactamase gene. The sequences of the detection probes were designed to match that of blaSHV-5 (mutation-prone codons for amino acids 179, 238, and 240 are highlighted in boldface type). The detection probe spanning the mutation site at position 179 was labeled at its 5′ end with LightCycler Red 640 dye and phosphorylated at its 3′ end to block extension. The corresponding anchor carried a fluorescein label (FITC) at its 3′ end. The detection probe covering positions 238 and 240 simultaneously was labeled at its 3′ end with FITC. The corresponding anchor was labeled at its 5′ end with LightCycler Red 705 dye and was phosphorylated at its 3′ end to block extension. The use of two acceptor fluorophores, LightCycler Red 640 and LightCycler Red 705, allowed simultaneous detection of all three important mutations in a single reaction in one tube.