The techniques of PCR and RT-PCR offer several advantages when compared to traditional viral diagnostic techniques, especially in terms of analytical sensitivity and time for assay completion. Unfortunately the high level of sensitivity, which approaches the single molecule level, also makes this technique prone to false positive results. Amplicon generated by previous positives reactions in which dUTP was substituted for dTTP can be degraded by UNG and thus theoretically eliminated as a source of template that would cause false positives in PCR or RT-PCR. UNG degrades contaminating uracil-containing DNA while leaving the natural, thymine-containing DNA intact. The precise, reproducible quantification of real-time RT-PCR provides a rapid method to assess and optimize the use of UNG to eliminate or reduce the impact of amplicon DNA in RT-PCR as well as determine the effects of UNG on RNA detection. The manufacturers' recommendation for the use of heat-labile UNG include addition of 1 unit enzyme per 50 μl reaction followed by an incubation time of 10 min at 15 – 25°C. In this study, this enzyme concentration and the incubation conditions were not found to eliminate amplicon DNA, presumably due at least in part to the short length of amplicon DNA (114 bases). To consistently degrade approximately 500 copies of amplicon DNA, a two-fold increase in UNG concentration was necessary. However, this concentration of UNG resulted in a nearly two cycle increase to reach the threshold for RNA detection. These results are corroborated by a recent publication using real-time PCR for detection of single-copy genes in which it was concluded that manufacturers' recommended conditions were not adequate to consistently degrade even minimal (e.g. < 30 copies) amounts of amplicon DNA .
To determine optimal conditions for amplicon degradation with minimal effect on RNA detection by RT-PCR, a series of time, temperature and enzyme concentrations were evaluated. From these results, it was shown that UNG degradation of amplicon DNA in RT-PCR was concentration, time and temperature depended, as previously described for real-time PCR . To allow degradation of considerable concentrations of carry-over amplicon DNA while having minimal effects on RNA detection, the optimal concentration of UNG was found to be 0.25 U per 25 μl reaction with a pre-amplification incubation at 30°C for 20 min. This incubation was performed after the complete reaction had been assembled in the reaction tubes, just prior to the RT step. These conditions completely degraded approximately 2,500 copies of carry-over amplicon DNA while reducing the detectible concentration of viral RNA template by 2.2-fold (i.e. CT values increased by a mean of 1.2 cycles). If higher levels of carry-over contamination are encountered, increasing the incubation time by an additional 10 min demonstrated degradation of a 10-fold higher concentration of amplicon DNA while only slightly increasing the CT for RNA detection. It is interesting to note that, based on analysis of the standard curves, increases observed in the CT for RNA were due both to the presence of UNG and the incubation at 30°C prior to RT-PCR.
Given the relatively long time required for the reverse transcriptase step, a heat-labile UNG that is rapidly and effectively inactivated at temperatures below that of the RT step must be used for this approach to be applied to control false positive reactions in RT-PCR. The commercially available heat-labile UNG used in this study is rapidly inactivated at 40°C with a half-life of 2 min . At the end of the UNG incubation, the samples were held at 55°C to rapidly inactivate UNG just prior to the RT step which was 50°C for 30 min. For the RT-PCR reagents used (as well as many other commercially availably RT-PCR reagents), the manufacture states that the 30 min RT step can be performed at temperatures of up to 55°C (or higher for some reagent systems), and reactions analyzed in preliminary experiments (data not shown) demonstrated no effect on RT-PCR analytical sensitivity following a 2 min incubation at 55°C prior to the RT step.
Results presented herein demonstrated that incubation with UNG appears to increase the CT equally for in vitro transcribed RNA and viral RNA, thus quantification through the use of a standard curve can remain accurate provided that all reactions are performed under the same conditions. However, a small percentage of weak positive samples may not be detected due to an increase in CT caused by UNG and incubation prior to RT-PCR. To the reduce the possibility of false negatives with dilute RNA samples, results from this study suggest that replicates of two or more reactions should be sufficient, given that the false negative rate was only increased by 2% for samples containing UNG and incubated prior to RT-PCR. Also, two – five additional cycles of amplification may provide positive amplification of target RNA in samples with very low concentrations.