FIG. 2.

Detection of bacteriophage MS2 genomic RNA by real-time fluorogenic RT-PCR. Thick horizontal lines represent the threshold (t) for significant signal detection. Larger amounts of RNA permitted positive detection (signal above threshold) with fewer PCR cycles. The greatest amount of RNA (400 pg) produced a signal with the fewest cycles (arrows). (A) Limit of detection experiments demonstrating the sensitivity of assay MS2-1, performed using 10-fold dilutions of a preparation of bacteriophage MS2 RNA (concentrations at right). Using the probe and primer set in assay 1 (numbered as in Table 1), 4 fg of RNA was repeatably and reproducibly detected. Assay MS-1 did not detect 0.4 fg of MS2 RNA: the assay gave results indistinguishable from the negative (no-template) control (NTC). (B) Cross-reactivity experiments using primers and probe from assay MS2-1. None of the non-target nucleic acid (NTNA) samples were amplified above threshold by the MS2-1 primer set, while 400 pg of MS2 RNA gave a positive reaction. ΔRn, change in fluorescence.