All real-time PCR methods in the Auburn University Molecular Diagnostics Laboratory are validated for robust amplification of single copies of target nucleic acid in a high background of unrelated nucleic acids.
Robust real-time PCR methodology for reliable amplification of single target copies. Three aliquots of nucleic acids extracted from EDTA blood of a dog chronically infected with very low numbers of Hepatozoon americanum are examined by amplification of Hepatozoon spp. 18S rRNA gene target sequences. The graph displays fluorescence intensity at each amplification cycle. During the exponential amplification phase, low target copy numbers are correlated with delayed appearance of the signal. The robustness of the assay is indicated by the fact that samples with 1 or 3 target copies display as efficient amplification as the standards with high copy numbers while samples without Hepatozoon americanum target DNA show only a background signal below the detection threshold.