Figure 3

Rapid amplification and detection of Neisseria gonorrhea genomic DNA. Panel A) Fluorescence monitoring in real-time is shown as semi-log plot of fluorescence intensity versus Tt number where each cycle is 1 minute-long. The copy numbers of input template are labeled on the figure. Panel B) Type II BESt™ cassette detection of 5e4 copies/assay (strips 1-2), 500 copies/assay (strips 3-4), 50 copies/assay (strips 5-7), and non template control (NTC) (strips 8-9). The signal from NTC (either in panel A or B) is the amplification signal from the internal control sequence. The IC sequence shares the same primer sequence with the detected target gene of Neisseria gonorrhoeae, just with different internal nucleic acid sequence between the primers. The IC sequence is cloned into a plasmid. When a reaction is set-up, the IC plasmid is premixed with all of the other reaction components (except the target DNA or tested clinical samples) to prepare the master mix of the assay. When there is no target template of Neisseria gonorrhoeae (or negative clinical samples) in the assay, the IC sequence is amplified. Because the different internal sequence between the IC sequence and the target sequence of Neisseria gonorrhoeae, different probes are designed and included in the reaction. This can be differentiated from T line (Test line: for the detection of target DNA amplification products by target probe NGP) and C line (Control line: for the detection of internal control amplification products by IC probe NGICP) in Panel B.