Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection

Abstract

Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

Conflict of interest statement

Competing Interests: RS is an inventor on patents related to recombinant AAV technology. RS owns equity in a gene therapy company that is commercializing AAV for gene therapy applications. To the extent that the work in this manuscript increases the value of these commercial holdings, RS has a conflict of interest. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. The other authors have no conflicts of interest.

Figures

Figure 1. Internal Threshold Control (ITC) assay format.

Panel (A) ITCEPO duplex assay format. The EPO probe specifically detects the EPO cDNA harbored in rAAV vectors and does not bind to the genomic DNA because it rests on an exon/exon junction. The EPO ITC probe recognizes only the synthetic ITCEPO template. The EPO primers recognize the EPO cDNA, the ITCEPO template, and the genomic EPO locus. Panel (B) ITCCMV duplex assay format. The CMV probe specifically detects the CMV immediate early promoter region in the pSSV9-MD2-cmEPO plasmid. The CMV ITC probe recognizes only the synthetic ITCCMV template. The CMV primers recognize the CMV promoter and the ITCCMV template.

Figure 2. ITC duplex assay equivalence testing.

All three ITC duplex assays were tested in the presence of same amount of target and ITC template and 500 ng naïve gDNA. Five copies of pSSV9-MD2-cmEPO plasmid and ITCcmEPO template were amplified using the ITCcmEPO duplex assay. 10 copies of pShuttle-CAG-hEPO-pA plasmid and ITChEPO template were amplified using the ITChEPO duplex assay. 10 copies of pSSV9-MD2-cmEPO plasmid and ITCCMV template were amplified using the ITCCMV duplex assay. Each reaction was repeated 15 times. Similarities in mean Ct values were analyzed statistically as described in Materials and Methods.

Figure 3. ITC duplex assay competition testing.

The copy number of the ITC template was held at 5 copies (cmEPO ITC) or 10 copies (hEPO or CMV ITC) in each reaction, while the target template was titrated from 5 or 10 copies to 100 copies. Each reaction was repeated 5 times in the presence of 500 ng naïve gDNA to obtain the mean Ct value. Panel (A) ITCcmEPO duplex assay, Panel (B) ITChEPO duplex assay and Panel (C) ITCCMV duplex assay.

Figure 4. ITC duplex assay interference testing.

Target plasmid (♦) and ITC template (O) were amplified in one reaction in the presence of 500 ng naïve gDNA to test the influence on the Ct between the two DNA targets. The total copy number of the plasmid and ITC template was maintained at 100. The target plasmid copy number is 10, 25, 50, 75, 90 and the ITC template copy number is 90, 75, 50, 25, 10 from left to right. Each reaction was repeated 5 times to obtain the mean Ct value. Panel (A) ITCcmEPO duplex assay Panel (B) ITChEPO duplex assay and Panel (C) ITCCMV duplex assay.